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  • 1.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andresen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Khan, Shahid N.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Kay, Lewis E.
    Departments of Medical Genetics, Biochemistry and Chemistry, The University of Toronto, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity2015In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 3, p. 341-351Article in journal (Refereed)
    Abstract [en]

    A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.

  • 2.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Carlsson, Mats
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    PINT: a software for integration of peak volumes and extraction of relaxation rates2013In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 56, no 3, p. 191-202Article in journal (Refereed)
    Abstract [en]

    We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.

  • 3.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Khan, Shahid N.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Forman-Kay, Julie D.
    Molecular Structure and Function Program, Hospital for Sick Children; Department and Biochemistry, University of Toronto, Canada.
    Sicheri, Frank
    cDepartment and Biochemistry, University of Toronto; Department of Molecular Genetics, University of Toronto; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase DomainManuscript (preprint) (Other academic)
    Abstract [en]

    Active and autoinhibited forms of the ephrinB receptor 2 (EphB2) kinase domain have been studied using NMR spectroscopy. The project was initiated because of the finding that the crystal structures of active forms of the kinase domain and previous NMR studies suggested that a change in inter-lobe flexibility and the sampling of catalytically competent excited states conformations are responsible for activity. Using Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, we have measured millisecond dynamics to identify such states. We have also performed concentration dependent relaxation experiments and analytical ultracentrifugation experiments that report on the effective protein size to look for possible differences in self-association for active and autoinhibited forms of the EphB2 kinase domain. We show that the active but not autoinhibited forms exchange between a ground state and an excited state at a rate of 1900 s-1. Similar results were found for the S677/680A mutant of the protein. The nature and importance of the excited state is still unknown. Our most important finding is that active forms of the kinase domain self-associate in a concentration dependent manner and form tetramers and possibly larger oligomers. Multimerization of the kinase domain may enable the assembly of complexes of downstream proteins and could be important for Eph signaling.

  • 4.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mayzel, Maxim
    The Swedish NMR Centre, University of Gothenburg, Sweden.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Orekhov, Vladislav Y.
    The Swedish NMR Centre, University of Gothenburg, Sweden.
    Measurement of Protein Backbone 13CO and 15N Relaxation Dispersion at High ResolutionManuscript (preprint) (Other academic)
    Abstract [en]

    Three-dimensional pulse sequences for the measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions and new methods for co-processing non-uniformly sampled data are presented. The new methodology was validated for the disordered protein IgA and for an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that the results are similar to ones obtained using traditional experiments and that accurate excited state chemical shifts can be determined. Furthermore, we show that jackknife analysis of down sampled spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points. The methodology should be useful for characterization of millisecond dynamics in small to medium-sized proteins with poorly dispersed spectra.

  • 5.
    Akke, Mikael
    et al.
    Department of Biophysical Chemistry, Lund University.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Conformational Dynamics by Relaxation Dispersion2012In: Encyclopedia of Biophysics / [ed] Gordon C. K. Roberts., Elsevier, 2012Chapter in book (Other academic)
    Abstract [en]

    The Encyclopedia of Biophysics is envisioned both as an easily accessible source of information and as an introductory guide to the scientific literature. It includes entries describing both Techniques and Systems. In the Techniques entries, each of the wide range of methods which fall under the heading of Biophysics are explained in detail, together with the value and the limitations of the information each provides. Techniques covered range from diffraction (X-ray, electron and neutron) through a wide range of spectroscopic methods (X-ray, optical, EPR, NMR) to imaging (from electron microscopy to live cell imaging and MRI), as well as computational and simulation approaches.In the Systems entries, biophysical approaches to specific biological systems or problems – from protein and nucleic acid structure to membranes, ion channels and receptors – are described. These sections, which place emphasis on the integration of the different techniques, therefore provide an inroad into Biophysics from a biological more than from a technique-oriented physical/chemical perspective. Thus the Encyclopedia is intended to provide a resource both for biophysicists interested in methods beyond those used in their immediate sub-discipline and for those readers who are approaching biophysics from either a physical or biological background.

  • 6.
    Anandapadmanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Helander, Sara
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Ohyama, Yoshifumi
    Yokohama City University, Japan .
    Siponen, Marina I.
    Karolinska Institute, Sweden .
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Kokubo, Tetsuro
    Yokohama City University, Japan .
    Ikura, Mitsuhiko
    University of Toronto, Canada .
    Moche, Martin
    Karolinska Institute, Sweden .
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation2013In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 20, no 8, p. 1008-+Article in journal (Refereed)
    Abstract [en]

    The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

  • 7.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Helander, Sara
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lemak, Alexander
    University of Toronto, Canada .
    Fares, Christophe
    University of Toronto, Canada .
    Csizmok, Veronika
    Hospital for Sick Children, Canada .
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Penn, Linda Z
    University of Toronto, Canada .
    Forman-Kay, Julie D
    Hospital Sick Children, Canada University of Toronto, Canada .
    Arrowsmith, Cheryl H
    University of Toronto, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, p. 6353-6366Article in journal (Refereed)
    Abstract [en]

    The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

  • 8.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Helander, Sara
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Lemak, Alexander
    Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
    Farès, Christophe
    Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
    Csizmok, Veronika
    Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Penn, Linda Z.
    Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
    Forman-Kay, Julie D.
    Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
    Arrowsmith, Cheryl H.
    Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Transient structure and intrinsic disorder in the c-Myc transactivation domain and its effects on ligand bindingManuscript (preprint) (Other academic)
    Abstract [en]

    The crucial role of c-Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of c-Myc function. The transactivation domain of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of c-Myc-1-88, where hierarchical phosphorylation of T58 and S62 regulates activation and destruction of the c-Myc protein. By NMR chemical shift analysis, relaxation measurements and NOE analysis, we show that both the MBI region (residues 45-65) and residues 22-33 are transiently structured regions, conserved also in other members of the Myc family. Binding of Bin1-SH3 to c-Myc-1-88 as assayed by NMR and SPR revealed primary binding to the S62 region, but also a dynamically disordered and multivalent complex in which intrinsic disorder of c-Myc-1-88 was retained while releasing transient intramolecular interactions. Our findings describe a novel mode of regulatory recognition of c-Myc that is in agreement with the increasingly recognized capability of intrinsically disordered regions to efficiently mediate transient interactions with a wide range of targets, with important implications towards understanding the unique multifaceted biological functions of c-Myc.

  • 9.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Structural and dynamic analysis of human glutaredoxin 3Manuscript (preprint) (Other academic)
    Abstract [en]

    Human glutaredoxin (Grx3) is an essential protein associated with biological functions including embryonic development and immune response, and is involved in human disease such as lung, colon cancer and cardiovascular disorder. Grx3 can harbour a [2Fe-2S]2+ cluster and is most likely involved in oxidative stress response. Grx3 consists of an N-terminal thioredoxin-like domain and two additional monothiol glutaredoxin domains, and is thus classified as a multidomain monothiol glutaredoxin. The Grx3 thioredoxin domain lacks both a characteristic active-site and catalytic activity, but is still essential in the yeast homologue and presumably functions together with its monothiol glutaredoxin domains. We have characterised the structures of the two Nterminal domains in Grx3, which have thioredoxin and glutaredoxin folds. We have analysed their dynamic and structural interdependence by analysing NMR relaxation data together with chemical shift changes between isolated and covalently linked domains. We find that although the two domains show interdomain mobility around a semi-flexible linker, there are indications for a preferred interaction surface between the two domains. Millisecond internal dynamics in a suggested ligand binding site in the isolated thioredoxin domain is dampened in the domain pair, suggesting that the two domains mutually affect each other on a profound level. Our results present a platform for further detailed studies of multidomain thioredoxin-glutaredoxin containing proteins, and their function in human cells.

  • 10.
    Auer, Renate
    et al.
    University of Toronto.
    Neudecker, Philipp
    University of Toronto.
    Muhandiram, D Ranjith
    University of Toronto.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Flemming Hansen, D
    University of Toronto.
    Konrat, Robert
    University of Vienna.
    Kay, Lewis E
    University of Toronto.
    Measuring the Signs of H-1(alpha) Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R-1 rho NMR Spectroscopy2009In: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ISSN 0002-7863, Vol. 131, no 31, p. 10832-10833Article in journal (Refereed)
    Abstract [en]

    Analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical, shift differences between NMR probes in the exchanging states, vertical bar Delta(pi)vertical bar, are also extracted. Herein, we present a simple experiment for obtaining the sign of H-1(alpha) Delta(pi) values by measuring off-resonance H-1(alpha) decay rates, R-1 rho, using weak proton spin-lock fields. A pair of R-1 rho values is measured with a spin-lock field applied vertical bar Delta omega vertical bar downfield and upfield of the major-state peak. In many cases, these two relaxation rates differ substantially, with the larger one corresponding to the case where the spin-lock field coincides with the resonance frequency of the probe in the minor state. The utility of the methodology is demonstrated first on a system involving protein ligand exchange and subsequently on an SH3 domain exchanging between a folded state and its on-pathway folding intermediate. With this experiment, it thus becomes possible to determine H-1(alpha) chemical shifts of the invisible excited state, which can be used as powerful restraints in defining the structural properties of these elusive conformers.

  • 11.
    Bach, Anders
    et al.
    University of Copenhagen.
    Clausen, Bettina H
    University of South Denmark.
    Moller, Magda
    University of Copenhagen.
    Vestergaard, Bente
    University of Copenhagen.
    Chi, Celestine N
    Uppsala University.
    Round, Adam
    European Molecular Biol Lab.
    Sorensen, Pernille L
    University of Copenhagen.
    Nissen, Klaus B
    University of Copenhagen.
    Kastrup, Jette S
    University of Copenhagen.
    Gajhede, Michael
    University of Copenhagen.
    Jemth, Per
    Uppsala University.
    Kristensen, Anders S
    University of Copenhagen.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Lambertsen, Kate L
    University of South Denmark.
    Stromgaard, Kristian
    University of Copenhagen.
    A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 9, p. 3317-3322Article in journal (Refereed)
    Abstract [en]

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

  • 12.
    Björnsson, Jon Mar
    et al.
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Andersson, Elisabet
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Lundström, Patrik
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Larsson, Nina
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Xu, Xiufeng
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Repetowska, Ewa
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Humphries, R. Keith
    Terry Fox Laboratory, British Columbia Cancer Agency.
    Karlsson, Stefan
    Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University.
    Proliferation of primitive myeloid progenitors can be reversibly  induced by HOXA102001In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 98, no 12, p. 3301-3308Article in journal (Refereed)
    Abstract [en]

    Recent studies show that several Hox transcription factors are important for regulation of proliferation and differentiation in hematopoiesis. Among these is H0XA10, which is selectively expressed at high levels in the most primitive subpopulation of human CD34+ bone marrow cells. When overexpressed, H0XA10 increases the proliferation of early progenitor cells and can lead to the development of myeloid leukemia. To study the effects of H0XA10 on primitive hematopoietic progenitors in more detail, transgenic mice were generated with regulatable H0XA10 expression. The transgenic mouse model, referred to astetO-HOXA10, contains theH0XA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of H0XA10 is inducible and reversible depending on the absence or presence of tetracycline or its analog, doxycycline. A retroviral vector containing the tetracycline transactivator gene (tTA) was used to induce expression of the H0XA10 gene in bone marrow cells from the transgenic mice. Reverse transcription–polymerase chain reaction analysis confirmed regulatable H0XA10 expression in several transgenic lines. H0XA10 induction led to the formation of hematopoietic colonies containing blastlike cells and megakaryocytes. Moreover, the induction of H0XA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFUS12]), which was reversible on withdrawal of induction. Activation of H0XA10 expression in tet0-H0XA10 mice will therefore govern proliferation of primitive myeloid progenitors in a regulated fashion. This novel animal model can be used to identify the target genes of HOXA10 and better clarify the specific role of HOXA10 in normal and malignant hematopoiesis.

  • 13.
    Björnsson, Jon Mar
    et al.
    Lund University, Department of Molecular Medicine.
    Larsson, Nina
    Lund University, Department of Molecular Medicine.
    Brun, Ann C. M.
    Lund University, Department of Molecular Medicine.
    Andersson, Elisabet
    Lund University, Department of Molecular Medicine.
    Lundström, Patrik
    Lund University, Department of Molecular Medicine.
    Larsson, Jonas
    Lund University, Department of Molecular Medicine.
    Repetowska, Ewa
    Lund University, Department of Molecular Medicine.
    Ehinger, Mats
    Lund University, Department of Molecular Medicine.
    Humphries, R. Keith
    University of British Columbia, Department of Medicine.
    Karlsson, Stefan
    Lund University, Department of Molecular Medicine.
    Reduced proliferative capacity of hematopoietic stem cells deficient in hoxb3 and hoxb42003In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 23, no 11, p. 3872-3883Article in journal (Refereed)
    Abstract [en]

    Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin(-) Scal(+) c-kit(+) hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.

  • 14.
    Chi, Celestine N
    et al.
    University of Copenhagen.
    Engstrom, Ake
    University of Copenhagen.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Ferguson, Neil
    University College Dublin.
    Jemth, Per
    Uppsala University.
    Biophysical Characterization of the Complex between Human Papillomavirus E6 Protein and Synapse-associated Protein 972011In: JOURNAL OF BIOLOGICAL CHEMISTRY, ISSN 0021-9258, Vol. 286, no 5, p. 3597-3606Article in journal (Refereed)
    Abstract [en]

    The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ(2) domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.

  • 15.
    Chi, Celestine N.
    et al.
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Haq, S. Raza
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Rinaldo, Serena
    University of Rome, Italy.
    Dogan, Jakob
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Cutruzzolà, Francesca
    University of Rome, Italy.
    Engström, Åke
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Gianni, Stefano
    University of Rome, Italy.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jemth, Per
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 44, p. 8971-8979Article in journal (Refereed)
    Abstract [en]

    The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.

  • 16.
    Ghasriani, Houman
    et al.
    University of Ottawa.
    Ducat, Thierry
    University of Ottawa.
    Hart, Chris T
    University of Ottawa.
    Hafizi, Fatima
    University of Ottawa.
    Chang, Nina
    University of Ottawa.
    Al-Baldawi, Ali
    University of Ottawa.
    Ayed, Saud H
    University of Ottawa.
    Lundström, Patrik
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Dillon, Jo-Anne R
    University of Saskatchewan.
    Goto, Natalie K
    University of Ottawa.
    Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE2010In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, ISSN 0027-8424, Vol. 107, no 43, p. 18416-18421Article in journal (Refereed)
    Abstract [en]

    MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first similar to 30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae, with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for full-length and N-terminal peptides from MinE demonstrated that residues that are buried in the dimeric interface nonetheless participate in direct interactions with MinD. According to results from NMR spin relaxation experiments, access to these buried residues may be facilitated by the presence of conformational exchange. We suggest that this concealment of MinD-binding residues by the MinE dimeric interface provides a mechanism for prevention of nonspecific interactions, particularly with the lipid membrane, to allow the free diffusion of MinE that is critical for Min protein oscillation.

  • 17.
    Hansen, Alexandar L
    et al.
    University of Toronto.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Velyvis, Algirdas
    University of Toronto.
    Kay, Lewis E
    University of Toronto.
    Quantifying Millisecond Exchange Dynamics in Proteins by CPMG Relaxation Dispersion NMR Using Side-Chain H-1 Probes2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 6, p. 3178-3189Article in journal (Refereed)
    Abstract [en]

    A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain H-1 positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {C-13,H-1}-glucose and D2O a significant number of isolated side-chain H-1 spins are generated, eliminating such effects. It thus becomes possible to record H-1 dispersion profiles at the beta positions of Ass, Cys, Ser, His, Phe, Tyr, and Trp as well as the gamma positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single H-1 dispersion experiment to 16. The utility of the approach is demonstrated with an application to the four-helix bundle colicin E7 immunity protein, Im7, which folds via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. The extracted H-1 chemical shift differences at side-chain positions provide valuable restraints in structural studies of invisible, excited states, complementing backbone chemical shifts that are available from existing relaxation dispersion experiments.

  • 18.
    Hansen, D. Flemming
    et al.
    University of Toronto, Canada.
    Vallurupalli, Pramodh
    University of Toronto, Canada.
    Lundström, Patrik
    University of Toronto, Canada.
    Neudecker, Philipp
    University of Toronto, Canada.
    Kay, Lewis E.
    University of Toronto, Canada.
    Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: How well can we do?2008In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 8, p. 2667-2675Article in journal (Refereed)
    Abstract [en]

    Carr−Purcell−Meiboom−Gill relaxation dispersion NMR spectroscopy has evolved into a powerful approach for the study of low populated, invisible conformations of biological molecules. One of the powerful features of the experiment is that chemical shift differences between the exchanging conformers can be obtained, providing structural information about invisible excited states. Through the development of new labeling approaches and NMR experiments it is now possible to measure backbone 13Cα and 13CO relaxation dispersion profiles in proteins without complications from 13C−13C couplings. Such measurements are presented here, along with those that probe exchange using 15N and 1HN nuclei. A key experimental design has been the choice of an exchanging system where excited-state chemical shifts were known from independent measurement. Thus it is possible to evaluate quantitatively the accuracy of chemical shift differences obtained in dispersion experiments and to establish that in general very accurate values can be obtained. The experimental work is supplemented by computations that suggest that similarly accurate shifts can be measured in many cases for systems with exchange rates and populations that fall within the range of those that can be quantified by relaxation dispersion. The accuracy of the extracted chemical shifts opens up the possibility of obtaining quantitative structural information of invisible states of the sort that is now available from chemical shifts recorded on ground states of proteins.

  • 19.
    Haq, S Raza
    et al.
    Uppsala University.
    Chi, Celestine N
    Uppsala University.
    Bach, Anders
    University of Copenhagen.
    Dogan, Jakob
    Uppsala University.
    Engstrom, Ake
    Uppsala University.
    Hultqvist, Greta
    Uppsala University.
    Karlsson, O Andreas
    Uppsala University.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Montemiglio, Linda C
    University of Roma La Sapienza.
    Stromgaard, Kristian
    University of Copenhagen.
    Gianni, Stefano
    University of Roma La Sapienza.
    Jemth, Per
    Uppsala University.
    Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, p. 599-605Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K-d correlate with changes in k(off).

  • 20.
    Helander, Sara
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Montecchio, Meri
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Pilstål, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Su, Yulong
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, USA.
    Kuruvilla, Jacob
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Sweden.
    Johansson, Malin
    Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Sweden.
    Mohammed, Javed
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Cristobal, Susana
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Sears, Rosalie
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, USA.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Department of Social and Welfare Studies, Learning, Aesthetics, Natural science. Linköping University, Faculty of Educational Sciences. Linköping University, Faculty of Science & Engineering.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Pre-Anchoring of Pin1 to Unphosphorylated c-Myc in a Fuzzy Complex Regulates c-Myc Activity2015In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, no 12, p. 2267-2279Article in journal (Refereed)
    Abstract [en]

    Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1–88 in the Pin1 interdomain cleft in a disordered, or “fuzzy”, complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1–88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells

  • 21.
    Hennig, Janosch
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Helmholtz Zentrum Munchen GmbH, Germany; Technical University of Munich, Germany.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Museth, Anna Katrine
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. CALTECH, CA 91125 USA.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Local Destabilization of the Metal-Binding Region in Human Copper-Zinc Superoxide Dismutase by Remote Mutations Is a Possible Determinant for Progression of ALS2015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 2, p. 323-333Article in journal (Refereed)
    Abstract [en]

    More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 degrees C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 degrees C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer beta-strands I and VI of the beta-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 degrees C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local beta-strands.

  • 22.
    Karlsson, Elin
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Magic, Ivana
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Bostner, Josefine
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Dyrager, Christine
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lysholm, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Hallbeck, Anna-Lotta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Stål, Olle
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Revealing Different Roles of the mTOR-Targets S6K1 and S6K2 in Breast Cancer by Expression Profiling and Structural Analysis2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 12, p. e0145013-Article in journal (Refereed)
    Abstract [en]

    Background

    The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer.

    Materials and methods

    Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1.

    Results

    Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.

  • 23.
    Korzhnev, Dmitry M.
    et al.
    University of Toronto, Ontario, Canada.
    Religa, Tomasz L.
    MRC, University of Cambridge, UK.
    Lundström, Patrik
    University of Toronto, ON, Canada.
    Fersht, Alan R.
    MRC, University of Cambridge, UK.
    Kay, Lewis E.
    University of Toronto, Ontario, Canada.
    The folding pathway of an FF domain: Characterization of an on-pathway intermediate state under folding conditions by N-15, C-13(alpha) and C-13-methyl relaxation dispersion and H-1/(2) H-exchange NMR Spectroscopy2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 2, p. 497-512Article in journal (Refereed)
    Abstract [en]

    The FF domain from the human protein HYPA/FBP11 folds via a lowenergy on-pathway intermediate (. Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by N-15, C-13(alpha) and methyl C-13 Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by 15N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U-I-F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone N-15, C-13(alpha) and side-chain methyl 13C chemical shifts, with further information from protection factors for the backbone amide groups from H-1/(2) H-exchange. Notably, helices H1-H3 are at least partially formed in 1, while helix H4 is largely disordered. Chemical shift differences for the methyl 13 C nuclei suggest a paucity of stable, native-like hydrophobic interactions in 1. These data are consistent with (D-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and (1) data can elucidate whole experimental folding pathways.

  • 24.
    Kukic, Predrag
    et al.
    University of Cambridge, England.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Camilloni, Carlo
    University of Cambridge, England.
    Evenas, Johan
    Red Glead Discovery, Sweden.
    Akke, Mikael
    Lund University, Sweden.
    Vendruscolo, Michele
    University of Cambridge, England.
    Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 1, p. 19-28Article in journal (Refereed)
    Abstract [en]

    Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca2+-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, N-15 order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca2+ by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca2+-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

  • 25.
    Lundström, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Blissing, Annica T.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Isotope labeling methods for large systems2012In: Isotope labeling in Biomolecular NMR / [ed] Hanudatta S. Atreya, Springer, 2012, p. 3-15Chapter in book (Refereed)
    Abstract [en]

    NMR spectroscopy has undergone a revolution in recent years with the advent of several new methods overcoming the problems of sensitivity and resolution. Recent developments in biotechnology have made it easier and economical to introduce 13C, 15N and 2H into proteins and nucleic acids. At the same time, there has been an explosion in the number of NMR experiments that utilize such isotope labeled samples. Thus, a combination of isotopic labeling and multidimensional, multinuclear NMR has opened up new avenues for structural studies of proteins, nucleic acids and their complexes. This book wil

  • 26.
    Lundström, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Blissing, Annica T.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Isotope labeling methods for relaxation measurements2012In: Isotope labeling in Biomolecular NMR / [ed] Hanudatta S. Atreya, Springer, 2012, p. 63-82Chapter in book (Refereed)
    Abstract [en]

    NMR spectroscopy has undergone a revolution in recent years with the advent of several new methods overcoming the problems of sensitivity and resolution. Recent developments in biotechnology have made it easier and economical to introduce 13C, 15N and 2H into proteins and nucleic acids. At the same time, there has been an explosion in the number of NMR experiments that utilize such isotope labeled samples. Thus, a combination of isotopic labeling and multidimensional, multinuclear NMR has opened up new avenues for structural studies of proteins, nucleic acids and their complexes. This book wil

  • 27.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Microsecond protein dynamics measured by C-13(alpha) rotating-frame spin relaxation2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 9, p. 1685-1692Article in journal (Refereed)
    Abstract [en]

    NMR spin relaxation in the rotating frame (R-1p) is a unique method for atomic-resolution characterisation of conformational (chemical) exchange processes occuring on the microsecond timescale. We present a rotating frame C-13 relaxation dispersion experiment for measuring conformational dynamics in uniformly C-13-labeled proteins. The experiment was validated by using the E140Q mutant of the C-terminal fragment of calmodulin, which exhibits significant conformational exchange between two major conformations, as gauged from previous N-15 and H-1 relaxation studies. Consistent with previous work, the present C-13, R-1p experiment detects conformational-exchange dynamics throughout the protein. The average correlation time of <tau(ex)> = 25 +/- 8 mu s is in excellent agreement with those determined previously from H-1 and N-15 R-1p, relaxation data: <tau(ex)> = 19 +/- 7 and 21 +/- 3 mu s, respectively. The extracted chemical-shift differences between the exchanging states reveal significant fluctuations in dihedral angles within single regions of Ramachandran phi-psi space that were not identified from the H-1 and N-15 relaxation data. The present results underscore the advantage of using several types of nuclei to probe exchange dynamics in biomolecules.

  • 28.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins2005In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 32, no 2, p. 163-173Article in journal (Refereed)
    Abstract [en]

    NMR spin relaxation in the rotating frame (R-1 rho) is a unique method for atomic-resolution characterization of conformational ( chemical) exchange processes occurring on the microsecond time scale. Here, we use amide H-1 off-resonance R-1 rho relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using N-15 or C-13 relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The H-1 off-resonance R-1 rho data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 mu s. This is in contrast to the case for N-15, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly N-15-enriched samples that were either highly enriched in H-2 or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the H-1 transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present H-1 off-resonance R-1 rho experiments are in excellent agreement with those determined previously using a combination of N-15 laboratory-frame and off-resonance R-1 rho relaxation data, with average values of <tau(ex)> = 19 +/- 7 and 21 +/- 3 mu s, respectively.

  • 29.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Quantitative analysis of conformational exchange contributions to H-1-N-15 multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, ISSN 0002-7863, Vol. 126, no 3, p. 928-935Article in journal (Refereed)
    Abstract [en]

    Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential H-1-N-15 multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the H-1 and N-15 chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential H-1-N-15 multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from N-15 off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the H-1 and N-15 spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the H-1 chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on H-1 single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.

  • 30.
    Lundström, Patrik
    et al.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Hansen, D. Flemming
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Kay, Lewis E.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively C-13 labeled samples2008In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 42, no 1, p. 35-47Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated C-13 spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (CO)-C-13 experiments however the large separation between (CO)-C-13 and C-13(alpha) chemical shifts offers hope that robust (CO)-C-13 dispersion profiles can be recorded on uniformly C-13 labeled samples, leading to the extraction of accurate (CO)-C-13 chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-C-13]-pyruvate and (NaHCO3,)-C-13 or uniformly labeled, generated from C-13-glucose. Very similar (CO)-C-13 chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

  • 31.
    Lundström, Patrik
    et al.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Hansen, D. Flemming
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Vallurupalli, Parmodh
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Kay, Lewis E.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Accurate Measurement of Alpha Proton Chemical Shifts of Excited Protein States by Relaxation Dispersion NMR Spectroscopy2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 5, p. 1915-1926Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy can provide detailed information about low populated, invisible states of protein molecules, including backbone chemical shifts of the invisible conformer and bond vector orientations that can be used as structural constraints. Notably, the measurement of H-1(alpha) chemical shifts in excited protein states has not been possible to date because, in the absence of suitable labeling, the homonuclear proton scalar coupling network in side chains of proteins leads to a significant degradation in the performance of proton-based relaxation dispersion experiments. Here we have overcome this problem through a labeling scheme in which proteins are prepared with U-H-2 glucose and 50% D2O/50% H2O that results in cleuteration levels of between 50-88% at the C-beta carbon. Effects from residual H-1(alpha)-H-1(beta) scalar couplings can be suppressed through a new NMR experiment that is presented here. The utility of the methodology is demonstrated on a ligand binding exchanging system and it is shown that H-1(alpha) chemical shifts extracted from dispersion profiles are, on average, accurate to 0.03 ppm, an order of magnitude better than they can be predicted from structure using a database approach. The ability to measure H-1(alpha) chemical shifts of invisible conformers is particularly important because such shifts are sensitive to both secondary and tertiary structure. Thus, the methodology presented is a valuable addition to a growing list of experiments for characterizing excited protein states that are difficult to study using the traditional techniques of structural biology.

  • 32.
    Lundström, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Lin, Hong
    Hospital for Sick Children, Toronto.
    Kay, Lewis E
    University of Toronto.
    Measuring C-13(beta) chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy2009In: JOURNAL OF BIOMOLECULAR NMR, ISSN 0925-2738, Vol. 44, no 3, p. 139-155Article in journal (Refereed)
    Abstract [en]

    A labeling scheme is introduced that facilitates the measurement of accurate C-13(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of C-13 enrichment (30-40%) at C-beta side-chain carbon positions for 15 of the amino acids with little C-13 label at positions one bond removed (a parts per thousand 5%). A pair of samples are produced using [1-C-13]-glucose/(NaHCO3)-C-12 or [2-C-13]-glucose as carbon sources with isolated and enriched (andgt; 30%) C-13(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of C-13(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.

  • 33.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Mulder, Frans A. A.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 102, no 47, p. 16984-16989Article in journal (Refereed)
    Abstract [en]

    Nuclear spin relaxation is a powerful method for studying molecular dynamics at atomic resolution. Recent methods development in biomolecular NMR spectroscopy has enabled detailed investigations of molecular dynamics that are critical for biological function, with prominent examples addressing allostery, enzyme catalysis, and protein folding. Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used C-13(alpha)(i - 1)/C-13(alpha)(i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i - 1 and i, i and i + 1, etc.), we identified correlated motions that extend through continuous segments of the sequence. We detected significant correlated conformational transitions in the native state of the E140Q mutant of the calmodulin C-terminal domain. Previous work has shown that this domain exchanges between two major conformational states that resemble the functionally relevant open and closed states of the WT protein, with a mean correlation time of approximate to 20 mu s. The present results reveal that an entire alpha-helix undergoes partial unraveling in a transient and cooperative manner.

  • 34.
    Lundström, Patrik
    et al.
    University of Toronto, ON, Canada.
    Teilum, Kaare
    Lund University, Sweden .
    Carstensen, Tommy
    Lund University, Sweden .
    Bezsonova, Irina
    University of Toronto, ON, Canada.
    Wiesner, Silke
    University of Toronto, ON, Canada.
    Hansen, D. Flemming
    University of Toronto, ON, Canada.
    Religa, Tomasz L.
    MRC, University of Cambridge, UK.
    Akke, Mikael
    Lund University, Sweden .
    Kay, Lewis E.
    University of Toronto, ON, Canada.
    Fractional C-13 enrichment of isolated carbons using [1-C-13]- or [2-C-13]-glucose facilitates the accurate measurement of dynamics at backbone C-alpha and side-chain methyl positions in proteins2007In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 38, no 3, p. 199-212Article in journal (Refereed)
    Abstract [en]

    A simple labeling approach is presented based on protein expression in [1-C-13]- or [2-C-13]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C-alpha sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta 1) are produced with isolated C-13 spins (i.e., no C-13-C-13 one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C-beta positions for 17 of the common 20 amino acids and there are no cases for which C-13(alpha)-(CO)-C-13 spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone N-15 studies. The utility of the labeling is established by recording C-13 R-1 rho and CPMG-based experiments on a number of different protein systems.

  • 35.
    Lundström, Patrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Vallurupalli, Pramodh
    University of Toronto.
    Hansen, D Flemming
    University of Toronto.
    Kay, Lewis E
    University of Toronto.
    Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy.2009In: Nature protocols, ISSN 1750-2799, Vol. 4, no 11, p. 1641-1648Article in journal (Refereed)
    Abstract [en]

    The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to millisecond conformational exchange processes. The samples necessary for an analysis of exchange dynamics are discussed, using the Abp1p SH3 domain from Saccharomyces cerevisiae as an example. For this system, the time frame for production of each sample, including in vitro refolding, is about 80 h. The samples so produced facilitate the measurement of accurate chemical shifts of low populated, invisible conformers that are part of the exchange pathway. The accuracy of the methodology has been established experimentally and the chemical shifts that are obtained provide important restraints in structure calculations of the excited state.

  • 36.
    Lundström, Patrik
    et al.
    University of Toronto, ON, Canada.
    Vallurupalli, Pramodh
    University of Toronto, ON, Canada.
    Religa, Tomasz
    MRC, University of Cambridge, UK.
    Dahlquist, Fredrick W.
    University of California at Santa Barbara, USA.
    Kay, Lewis E.
    University of Toronto, ON, Canada.
    A single-quantum methyl C-13-relaxation dispersion experiment with improved sensitivity2007In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 38, no 1, p. 79-88Article in journal (Refereed)
    Abstract [en]

    A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.

  • 37.
    Neudecker, Philipp
    et al.
    University of Toronto, Ontario, Canada.
    Lundström, Patrik
    University of Toronto, Ontario, Canada.
    Kay, Lewis E.
    University of Toronto, Ontario, Canada.
    Relaxation Dispersion NMR Spectroscopy as a Tool for Detailed Studies of Protein Folding2009In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 96, no 6, p. 2045-2054Article in journal (Refereed)
    Abstract [en]

    Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a "high-resolution" picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.

  • 38.
    Neudecker, Philipp
    et al.
    University of Toronto, Ontario, Canada .
    Robustellu, Paul
    University of Cambridge, UK.
    Cavalli, Andrea
    University of Cambridge, UK.
    Walsh, Patrick
    University of Toronto, Ontario, Canada .
    Lundström, Patrik
    University of Toronto, ON, Canada.
    Zarrine-Afsar, Arash
    University of Toronto, Ontario, Canada .
    Sharpe, Simon
    University of Toronto, Ontario, Canada .
    Vendruscolo, Michele
    University of Cambridge, UK.
    Kay, Lewis E.
    University of Toronto, Ontario, Canada .
    Structure of an Intermediate State in Protein Folding and Aggregation2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 336, no 5079, p. 362-366Article in journal (Refereed)
    Abstract [en]

    Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal beta strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.

  • 39.
    Niklasson, Markus
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Marsh, Joseph A.
    University of Edinburgh, Scotland.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches2015In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 11, no 1, p. e1004022-Article in journal (Refereed)
    Abstract [en]

    The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

  • 40.
    Niklasson, Markus
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Helander, Sara
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Roth, Marie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Zimdahl Kahlin, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Robust and convenient analysis of protein thermal and chemical stability2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, no 12, p. 2055-2062Article in journal (Refereed)
    Abstract [en]

    We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

  • 41.
    Niklasson, Markus
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Otten, Renee
    Howard Hughes Medical Institute and Department of Biochemistry, Brandeis University, Waltham, MA, USA..
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Schlagnitweit, Judith
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Petzold, Katja
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Comprehensive analysis of NMR data using advanced line shape fitting.2017In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 69, no 2, p. 93-99Article in journal (Refereed)
    Abstract [en]

    NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT ( https://pint-nmr.github.io/PINT/ ). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.

  • 42.
    Ravichandran, Ranjithkumar
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Islam, M. M.
    Karolinska Institute, Sweden; .
    Alarcon, E. I.
    University of Ottawa, Canada; .
    Samanta, Ayan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Wang, S.
    Uppsala University, Sweden.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hilborn, J.
    Uppsala University, Sweden.
    Griffith, May
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Phopase, Jaywant
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Functionalised type-I collagen as a hydrogel building block for bio-orthogonal tissue engineering applications2016In: Journal of materials chemistry. B, ISSN 2050-750X, E-ISSN 2050-7518, Vol. 4, no 2, p. 318-326Article in journal (Refereed)
    Abstract [en]

    In this study, we derivatized type I collagen without altering its triple helical conformation to allow for facile hydrogel formation via the Michael addition of thiols to methacrylates without the addition of other crosslinking agents. This method provides the flexibility needed for the fabrication of injectable hydrogels or pre-fabricated implantable scaffolds, using the same components by tuning the modulus from Pa to kPa. Enzymatic degradability of the hydrogels can also be easily fine-tuned by variation of the ratio and the type of the crosslinking component. The structural morphology reveals a lamellar structure mimicking native collagen fibrils. The versatility of this material is demonstrated by its use as a pre-fabricated substrate for culturing human corneal epithelial cells and as an injectable hydrogel for 3-D encapsulation of cardiac progenitor cells.

  • 43.
    Teilum, Kaare
    et al.
    Lund University, Department of Biophysical Chemistry.
    Brath, Ulrika
    Lund University, Department of Biophysical Chemistry.
    Lundström, Patrik
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Biosynthetic 13C labeling of aromatic side chains in proteins for NMR relaxation measurements2006In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, ISSN 0002-7863, Vol. 128, no 8, p. 2506-2507Article in journal (Refereed)
  • 44.
    Vallurupalli, Pramodh
    et al.
    University of Toronto, ON, Canada.
    Hansen, D. Flemming
    University of Toronto, ON, Canada.
    Lundström, Patrik
    University of Toronto, ON, Canada.
    Kay, Lewis E.
    University of Toronto, ON, Canada.
    CPMG relaxation dispersion NMR experiments measuring glycine H-1(alpha) and C-13(alpha) chemical shifts in the 'invisible' excited states of proteins2009In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 45, no 1-2, p. 45-55Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (> 0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of H-1(alpha) and C-13(alpha) chemical shifts of glycine residues in the excited state that reflects the fact that in this case the H-1(alpha), C-13(alpha) spins form a three-spin system that is more complex than the AX H-1(alpha)-C-13(alpha) spin systems in the other amino acids. Here pulse sequences for recording H-1(alpha) and C-13(alpha) CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which H-1(alpha), C-13(alpha) chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.

  • 45.
    Weininger, Ulrich
    et al.
    Lund University, Sweden .
    Blissing, Annica T.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Liu, Zhihong
    University of Calgary, Canada .
    Vogel, Hans J.
    University of Calgary, Canada .
    Akke, Mikael
    Lund University, Sweden .
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Protein conformational exchange measured by H-1 R-1 rho relaxation dispersion of methyl groups2013In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 57, no 1, p. 47-55Article in journal (Refereed)
    Abstract [en]

    Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for H-1 spins in methyl (CHD2)-C-13 groups, which improves the characterization of fast exchange processes. The influence of H-1-H-1 rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R (1 rho) relaxation data acquired with tilt angles of 90A degrees and 35A degrees, in which the ROE is maximal and minimal, respectively, and on samples containing different H-1 densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any H-1 relaxation dispersion of its methyl groups at 10 or 25 A degrees C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by H-1 and C-13 CPMG experiments. The present R (1 rho) experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

  • 46.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Blissing, Annica Theresia
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Partially Assigned Chemical Shifts of Human Thiopurine S-methyltransferase Reveal Flexibility in Native StructureManuscript (preprint) (Other academic)
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