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  • 1.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Bengt
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . 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.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, no 2, p. 619-633Article in journal (Refereed)
    Abstract [en]

    Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

  • 2.
    Bano-Polo, Manuel
    et al.
    University of Valencia, Spain .
    Martinez-Gill, Luis
    University of Valencia, Spain .
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Nieva, Jose L.
    University of Pais Vasco UPV EHU, Spain .
    Elofsson, Arne
    Stockholm University, Sweden .
    Mingarro, Ismael
    University of Valencia, Spain .
    Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion2013In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, no 4, p. 830-840Article in journal (Refereed)
    Abstract [en]

    alpha-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

  • 3.
    Brorsson, Ann-Christin
    et al.
    Department of Biochemistry, Umeå University.
    Kjellson, Annika
    Department of Biochemistry, Umeå University.
    Aronsson, Göran
    Biopool AB.
    Sethson, Ingmar
    Department of Organic Chemistry, Umeå University.
    Hambraeus, Charlotta
    University of Southern Stockholm.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    The "two-state folder" MerP forms partially unfolded structures that show temperature dependent hydrogen exchange2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 340, no 2, p. 333-344Article in journal (Refereed)
    Abstract [en]

    We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55°C. The temperature dependence of the hydrogen exchange has allowed us to determine ΔG, ΔH and ΔCp values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP. © 2004 Elsevier Ltd. All rights reserved.

  • 4.
    Brorsson, Ann-Christin
    et al.
    Department of Biochemistry, Umeå University.
    Lundqvist, Martin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Sethson, Ingmar
    Department of Organic Chemistry Umeå University.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 357, no 5, p. 1634-1646Article in journal (Refereed)
    Abstract [en]

    We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (ΔGHX) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions. © 2006 Elsevier Ltd. All rights reserved.

  • 5.
    Fladvad, Malin
    et al.
    Department of Medical Biochemistry and Biophysics Karolinska Institutet, Stockholm.
    Zhou, Kaison
    Molecular Biotechnology IFM, Linköping University.
    Moshref, Ahmed
    Department of Medical Biochemistry and Biophysics Karolinska Institutet, Stockholm.
    Pursglove, Sharon
    School of Molecular and Microbial Biosciences University of Sydney.
    Säfsten, Pär
    Biacore AB, Uppsala.
    Sunnerhagen, Maria
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    N and C-terminal Sub-regions in the c-Myc Transactivation Region and their Joint Role in Creating Versatility in Folding and Binding2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 246, p. 175-189Article in journal (Refereed)
  • 6.
    Hennig, Janosch
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Bresell, Anders
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Sandberg, Martina
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Hennig, Klaus D. M.
    Gabriele von Bülow Gymnasium, Berlin.
    Wahren-Herlenius, Marie
    Karolinska Hospital.
    Persson, Bengt
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    The fellowship of the RING: The RING-B-box linker region (RBL) interacts with the RING in TRIM21/Ro52, contributes to an autoantigenic epitope in Sjögren's syndrome, and is an integral and conserved region in TRIM proteins2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 377, no 2, p. 431-449Article in journal (Refereed)
    Abstract [en]

    Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn2+-binding region, which comprises an N-terminal RING followed by a B-box, separated by a ∼40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn2+ increases its stability. Regions stabilized by Zn2+ binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.

  • 7.
    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.

  • 8.
    Mishra, Rajesh
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Sörgjerd, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Nyström, Sofie
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Nordigården, Amanda
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Chiu, Yu-Jui
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lysozyme Amyloidogenesis Is Accelerated by Specific Nicking and Fragmentation but Decelerated by Intact Protein Binding and Conversion2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 366, no 3, p. 1029-1044Article in journal (Refereed)
    Abstract [en]

    We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 °C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently, however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth. © 2006 Elsevier Ltd. All rights reserved.

  • 9.
    Rantanen, V.-V.
    et al.
    Department of Mathematics, University of Turku, FIN-20014, Turku, Finland, Department of Biochemistry and Pharmacy, Åbo Akademi University, Tykistökatu 6 BioCity 3A, FIN-20521, Turku, Finland.
    Denessiouk, K.A.
    Department of Biochemistry and Pharmacy, Åbo Akademi University, Tykistökatu 6 BioCity 3A, FIN-20521, Turku, Finland.
    Gyllenberg, M.
    Department of Mathematics, University of Turku, FIN-20014, Turku, Finland.
    Koski, Timo
    Linköping University, The Institute of Technology. Linköping University, Department of Mathematics, Mathematical Statistics .
    Johnson, M.S.
    Department of Biochemistry and Pharmacy, Åbo Akademi University, Tykistökatu 6 BioCity 3A, FIN-20521, Turku, Finland.
    A fragment library based on Gaussian mixtures predicting favorable molecular interactions2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 313, no 1, p. 197-214Article in journal (Refereed)
    Abstract [en]

    Here, a protein atom-ligand fragment interaction library is described. The library is based on experimentally solved structures of protein-ligand and protein-protein complexes deposited in the Protein Data Bank (PDB) and it is able to characterize binding sites given a ligand structure suitable for a protein. A set of 30 ligand fragment types were defined to include three or more atoms in order to unambiguously define a frame of reference for interactions of ligand atoms with their receptor proteins. Interactions between ligand fragments and 24 classes of protein target atoms plus a water oxygen atom were collected and segregated according to type. The spatial distributions of individual fragment - target atom pairs were visually inspected in order to obtain rough-grained constraints on the interaction volumes. Data fulfilling these constraints were given as input to an iterative expectation-maximization algorithm that produces as output maximum likelihood estimates of the parameters of the finite Gaussian mixture models. Concepts of statistical pattern recognition and the resulting mixture model densities are used (i) to predict the detailed interactions between Chlorella virus DNA ligase and the adenine ring of its ligand and (ii) to evaluate the "error" in prediction for both the training and validation sets of protein-ligand interaction found in the PDB. These analyses demonstrate that this approach can successfully narrow down the possibilities for both the interacting protein atom type and its location relative to a ligand fragment. © 2001 Academic Press.

  • 10.
    Schutz, Patrick
    et al.
    Karolinska Institutet, Stockholm.
    Wahlberg, Elisabet
    Karolinska Institutet, Stockholm.
    Karlberg, Tobias
    Karolinska Institutet, Stockholm.
    Hammarström, Martin
    Karolinska Institutet, Stockholm.
    Collins, Ruairi
    Karolinska Institutet, Stockholm.
    Flores, Alex
    Karolinska Institutet, Stockholm.
    Schuler, Herwig
    Karolinska Institutet, Stockholm.
    Crystal structure of human RNA helicase A (DHX9: structural basis for unselective nucleotide base binding in a DEAD-box variant protein2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 400, no 4, p. 768-782Article in journal (Refereed)
    Abstract [en]

    RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long alpha-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5'-triphosphate, cytidine 5'-triphosphate, and uridine 5'-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.

  • 11. Ståhl, Annelie
    et al.
    Nilsson, Stefan
    Lundberg, Pontus
    Bhushan, Shashi
    Biverståhl, Henrik
    Moberg, Per
    Morisset, Magali
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Mäler, Lena
    Langel, Ulo
    Glaser, Elzbieta
    Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, no 4, p. 847-860Article in journal (Refereed)
    Abstract [en]

    Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F 1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P′1 position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent. © 2005 Elsevier Ltd. All rights reserved.

  • 12.
    Sörgjerd, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Ghafouri, Bijar
    Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Kelly, Jeffery W.
    The Skaggs Institute of Chemical Biology and the Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.
    Blond, Sylvie Y.
    Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago, IL, USA .
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Retention of Misfolded Mutant Transthyretin by the Chaperone BiP/GRP78 Mitigates Amyloidogenesi2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 356, no 2, p. 469-482 Article in journal (Refereed)
    Abstract [en]

    Carriers of the D18G transthyretin (TTR) mutation display an unusual central nervous system (CNS) phenotype with late onset of disease. D18G TTR is monomeric and highly prone to misfold and aggregate even at physiological conditions. Extremely low levels of mutant protein circulate both in human serum and cerebrospinal fluid, indicating impaired secretion of D18G TTR. Recent data show efficient selective ER-associated degradation (ERAD) of D18G TTR. One essential component of the ER-assisted folding machinery is the molecular chaperone BiP. Co-expression of BiP and D18G TTR, or BiP and wild-type (wt) TTR, or mutants A25T TTR and L55P TTR in Escherichia coli showed that only D18G TTR was significantly captured by BiP. Negligible capture of wt TTR and L55P TTR was seen and a sixfold smaller amount of A25T TTR bound to BiP compared to D18G TTR. These data correlate very well with thermodynamic and kinetic stability of the TTR variants, indicating that folding efficiency is inversely correlated to BiP capture. The complexes between BiP and D18G TTR were stable and could be isolated through affinity chromatography. Analytical ultracentrifugation and size-exclusion chromatography revealed that D18G TTR and BiP formed a mixture of 1:1 complexes and large soluble oligomers. The stoichiometry of captured D18G TTR versus BiP increased with increasing size of the oligomers. This indicates that BiP either worked as a molecular shepherd collecting the aggregation-prone mutant into stable oligomers or that BiP could bind to oligomers formed from misfolded mutant protein. Sequence analysis of bound TTR peptides to BiP revealed a bound sequence corresponding to residues 88–103 of TTR, comprising β-strand F in the folded TTR monomer constituting part of the hydrogen bonding tetramer interface in native TTR. The F-strand has also been suggested as a possible elongation region of amyloid fibrils, implicating how substoichiomeric amounts of BiP could sequester prefibrillar amyloidogenic oligomers through binding to this part of TTR. BiP binding to D18G TTR was abolished by addition of ATP. The released D18G TTR completely misfolded into amyloid aggregates as shown by ThT fluorescence and Congo red binding.

  • 13.
    Virkki, Minttu T.
    et al.
    Stockholm University, Sweden .
    Peters, Christoph
    Stockholm University, Sweden; Swedish e-Science Research Center (SeRC), Stockholm, Sweden.
    Nilsson, Daniel
    Stockholm University, Sweden .
    Sörensen, Therese
    Stockholm University, Sweden .
    Cristobal, Susana
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. University of the Basque Country, Leioa, Spain.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Elofsson, Arne
    Stockholm University, Sweden; Swedish E Science Research Centre SeRC, Stockholm, Sweden .
    The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 16, p. 2982-2991Article in journal (Refereed)
    Abstract [en]

    The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.

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