liu.seSearch for publications in DiVA
Change search
Refine search result
1 - 16 of 16
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Salomonsson, Johannes
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Sjöstrand, Linda
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
    D´arcy, Padraig
    Linköping University, Department of Biomedical and Clinical Sciences, Division of Clinical Chemistry and Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Transient interdomain interactions in free USP14 shape its conformational ensemble2024In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 33, no 5, article id e4975Article in journal (Refereed)
    Abstract [en]

    The deubiquitinase (DUB) ubiquitin-specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin-like (Ubl) domain separated by a 25-residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the proteasome, where the Ubl and USP domains of USP14 bind to the Rpn1 and Rpt1/Rpt2 units, respectively. No structure of full-length USP14 in the absence of proteasome has yet been presented, however, earlier work has described how transient interactions between Ubl and USP domains in USP4 and USP7 regulate DUB activity. To better understand the roles of the Ubl and USP domains in USP14, we studied the Ubl domain alone and in full-length USP14 by nuclear magnetic resonance spectroscopy and used small angle x-ray scattering and molecular modeling to visualize the entire USP14 protein ensemble. Jointly, our results show how transient interdomain interactions between the Ubl and USP domains of USP14 predispose its conformational ensemble for proteasome binding, which may have functional implications for proteasome regulation and may be exploited in the design of future USP14 inhibitors.

  • 2.
    Wei, Yong
    et al.
    Univ Hlth Network, Canada; Univ Toronto, Canada; Sunnybrook Res Inst, Canada.
    Redel, Cornelia
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lemak, Alexander
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Johansson-Åkhe, Isak
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Houliston, Scott
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Kenney, Tristan M. G.
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Tamachi, Aaliya
    Univ Hlth Network, Canada.
    Morad, Vivian
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Duan, Shili
    n/a.
    Andrews, David W.
    Sunnybrook Res Inst, Canada; Univ Toronto, Canada.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Arrowsmith, Cheryl H.
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Penn, Linda Z.
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    The MYC oncoprotein directly interacts with its chromatin cofactor PNUTS to recruit PP1 phosphatase2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 6, p. 3505-3522Article in journal (Refereed)
    Abstract [en]

    Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.

    Download full text (pdf)
    fulltext
  • 3.
    Selvaraj, Karthik
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Mofers, Arjan
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Pellegrini, Paola
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Salomonsson, Johannes
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Morad, Vivian
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hillert, Ellin-Kristina
    Karolinska Inst, Sweden.
    Espinosa, Belen
    Karolinska Inst, Sweden.
    Arner, Elias S. J.
    Karolinska Inst, Sweden.
    Jensen, Lasse
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology.
    Malmstrom, Jonas
    Recipharm AB, Sweden.
    Turkina, Maria V
    Linköping University, Department of Clinical and Experimental Medicine, Divison of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences.
    D´arcy, Padraig
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Walters, Michael A.
    Univ Minnesota, MN 55455 USA.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Linder, Stig
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Karolinska Inst, Sweden.
    Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 9841Article in journal (Refereed)
    Abstract [en]

    A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (similar to 20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.

    Download full text (pdf)
    fulltext
  • 4.
    Anandapadamanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Kyriakidis, Nikolaos C.
    Karolinska Univ Hosp, Sweden; UDLA, Ecuador.
    Csizmok, Veronika
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wallenhammar, Amélie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Espinosa, Alexander C.
    Karolinska Univ Hosp, Sweden.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Round, Adam R.
    Grenoble Outstn, France; European XFEL GmbH, Germany.
    Trewhella, Jill
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Univ Sydney, Australia.
    Moche, Martin
    Karolinska Inst, Sweden.
    Wahren-Herlenius, Marie
    Karolinska Univ Hosp, Sweden.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E22019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 294, no 30, p. 11404-11419Article in journal (Refereed)
    Abstract [en]

    The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-angstrom crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called linchpin residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.

  • 5.
    Wei, Yong
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering. Univ Hlth Network, Canada; Struct Genom Consortium, Canada.
    Resetca, Diana
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Li, Zhe
    Yokohama City Univ, Japan.
    Johansson-Åkhe, Isak
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Helander, Sara
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Chemistry. Linköping University, Faculty of Medicine and Health Sciences.
    Wallenhammar, Amélie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Morad, Vivian
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Raught, Brian
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Kokubo, Tetsuro
    Yokohama City Univ, Japan.
    Tong, Yufeng
    Struct Genom Consortium, Canada; Univ Windsor, Canada.
    Penn, Linda Z.
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Multiple direct interactions of TBP with the MYC oncoprotein2019In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 26, no 11, p. 1035-+Article in journal (Refereed)
    Abstract [en]

    Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -angstrom resolution crystal structure reveals that human MYC amino acids 98-111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1(TAND1)). Using biochemical approaches, we have shown that MYC amino acids 115-124 also interact with TBP independently of TAF1(TAND1). Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.

  • 6.
    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.

    Download full text (pdf)
    fulltext
  • 7.
    Mayzel, Maxim
    et al.
    University of Gothenburg, Sweden.
    Ahlner, Alexandra
    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.
    Orekhov, Vladislav Y.
    University of Gothenburg, Sweden.
    Measurement of protein backbone (CO)-C-13 and N-15 relaxation dispersion at high resolution2017In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 69, no 1Article in journal (Refereed)
    Abstract [en]

    Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points.

    Download full text (pdf)
    fulltext
  • 8.
    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.

    Download full text (pdf)
    fulltext
  • 9.
    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.

    Download full text (pdf)
    fulltext
  • 10. Order onlineBuy this publication >>
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins.

    To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals.

    To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism. 

    List of papers
    1. Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches
    Open this publication in new window or tab >>Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches
    Show others...
    2015 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 11, no 1, p. e1004022-Article in journal (Refereed) Published
    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.

    Place, publisher, year, edition, pages
    Public Library of Science, 2015
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-115010 (URN)10.1371/journal.pcbi.1004022 (DOI)000349309400013 ()25569628 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council [Dnr. 2012-5136]

    Available from: 2015-03-09 Created: 2015-03-06 Last updated: 2021-12-28
    2. PINT: a software for integration of peak volumes and extraction of relaxation rates
    Open this publication in new window or tab >>PINT: a software for integration of peak volumes and extraction of relaxation rates
    2013 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 56, no 3, p. 191-202Article in journal (Refereed) Published
    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.

    Place, publisher, year, edition, pages
    Springer Verlag (Germany), 2013
    Keywords
    Peak integration, Overlapped peaks, Relaxation rates, Protein dynamics
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:liu:diva-95951 (URN)10.1007/s10858-013-9737-7 (DOI)000321544600001 ()
    Note

    Funding Agencies|Swedish Research Council||

    Available from: 2013-08-19 Created: 2013-08-12 Last updated: 2021-12-28Bibliographically approved
    3. Measurement of Protein Backbone 13CO and 15N Relaxation Dispersion at High Resolution
    Open this publication in new window or tab >>Measurement of Protein Backbone 13CO and 15N Relaxation Dispersion at High Resolution
    (English)Manuscript (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.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-117070 (URN)
    Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2021-12-28Bibliographically approved
    4. Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity
    Open this publication in new window or tab >>Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity
    Show others...
    2015 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 3, p. 341-351Article in journal (Refereed) Published
    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.

    Place, publisher, year, edition, pages
    Springer Netherlands, 2015
    Keywords
    CPMG, 13Cα labeling, [2-13C]-Glycerol, Excited states
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-117073 (URN)10.1007/s10858-015-9948-1 (DOI)000357489200010 ()
    Note

    At the time for thesis presentation publication was in status: Manuscript

    At the time for thesis presentation name of publication was: Fractional enrichment using [2-13C]-glycerol as the carbon source facilitates measurements of excited state 13Cα chemical shifts with improved sensitivity

    Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2021-12-28Bibliographically approved
    5. Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase Domain
    Open this publication in new window or tab >>Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase Domain
    Show others...
    (English)Manuscript (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.

    Keywords
    Kinase activation | Eph receptors | chemical exchange | nmr spectroscopy | protein dynamics | self-association
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:liu:diva-117071 (URN)
    Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2021-12-28Bibliographically approved
    Download full text (pdf)
    fulltext
    Download (pdf)
    omslag
    Download (jpg)
    presentationsbild
  • 11.
    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.

    Download full text (pdf)
    fulltext
  • 12.
    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.

  • 13.
    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, Dordrecht: Springer, 2012, p. 3-15Chapter in book (Refereed)
    Abstract [en]

    A major drawback of nuclear magnetic resonance (NMR) spectroscopy compared to other methods is that the technique has been limited to relatively small molecules. However, in the last two decades the size limit has been pushed upwards considerably and it is now possible to use NMR spectroscopy for structure calculations of proteins of molecular weights approaching 100 kDa and to probe dynamics for supramolecular complexes of molecular weights in excess of 500 kDa. Instrumental for this progress has been development in instrumentation and pulse sequence design but also improved isotopic labeling schemes that lead to increased sensitivity as well as improved spectral resolution and simplification. These are described and discussed in this chapter, focusing on labeling schemes for amide proton and methyl proton detected experiments. We also discuss labeling methods for other potentially useful positions in proteins.

  • 14.
    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]

    Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynamics. Not only is it possible to use it for determination of time constant(s) for molecular reorientation but it can also be used to characterize internal motions on time scales from picoseconds to seconds. Traditionally, uniformly 15N labeled samples have been used for these experiments but it is clear that this limits the applications. For instance, sensitivity for large systems is dramatically increased if dynamics is probed at methyl groups and structural characterization of low-populated states requires measurements on 13Cα, 13Cβ or 13CO or 1Hα. Unfortunately, homonuclear scalar couplings may lead to artifacts in the latter types of experiments and selective isotopic labeling schemes that only label the desired position are necessary. Both selective and uniform labeling schemes for measurements of relaxation rates for a large number of positions in proteins are discussed in this chapter.

  • 15.
    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.

  • 16.
    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.

1 - 16 of 16
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf