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    Last Name

    Charles Robert Matthews PhD

    TitleChair and Professor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentBiochemistry and Molecular Pharmacology
    AddressUniversity of Massachusetts Medical School
    364 Plantation Street, LRB
    Worcester MA 01605
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentBiochemistry and Molecular Pharmacology

      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentBioinformatics and Computational Biology

      InstitutionUMMS - Programs, Centers and Institutes
      DepartmentBioinformatics and Integrative Biology

      InstitutionUMMS - Programs, Centers and Institutes
      DepartmentCenter for AIDS Research


        Academic Background

        B.S. University of Minnesota1968
        M.S. Stanford University1969
        Ph.D. Stanford University1974
        Post-Doctoral Fellow Stanford University1975
        Professor of Chemistry, The Pennsylvania State University1975-2000

        Arthur F.and Helen P. Koskinas Professor of Biochemistry
        and Molecular Pharmacology

        Solving the Protein Folding Problem

        Dr.  C.Robert Matthews

        Determining the mechanism by which the amino acid sequence of a protein directs the rapid and efficient folding of proteins to their native, functional forms remains one of the most challenging problems in molecular biophysics. The development of a folding code that specifies the three-dimensional structure adopted by a given sequence would complement the genetic code and complete the central dogma of molecular biology (DNA " RNA " Amino Acid Sequence" Functional Protein). Over the past decade, it has also become clear that failure to fold correctly and quickly can lead to numerous diseases. The goals of our lab are to obtain a molecular-level understanding of the folding mechanisms of several very common motifs and to use those insights to contribute to the development of a general theory of protein folding. More recently, we have become interested in applying these biophysical insights to the development of therapeutics for misfolding diseases.

        Ongoing projects in the lab

        (βα)8-barrel proteins
        Barrel Proteins

        The (βα)8-barrel, or TIM barrel motif, is the most commonly observed fold in biology. TIM barrels are found in five of the six classes of enzymes, and about 10% of all proteins with known three-dimensional structures contain at least one TIM barrel domain. The canonical (βα)8-barrel contains about 200-260 amino acids and is composed of eight βα-repeat units comprised of a β-strand and an anti-parallel α-helix connected by a βα-loop. The eight parallel β-strands form a cylindrical barrel that is surrounded by a shell of the α-helices. In all known (βα)8-barrels, the catalytic active residues are located at the C-terminal ends of the β-strands and in the βα-loops, while residues maintaining the stability of the fold are found at the opposite end of the barrel. The similar topology and structure observed for (βα)8-barrel proteins often arises from vastly divergent amino acid sequences. Thus, TIM barrel proteins are an excellent system for assessing the contributions of the topology and the sequence in governing the folding pathway and the stability of the native structure. Although topology dictates the basic features of the folding free energy surface, we have found that large clusters of isoleucine, leucine and valine side chains between the β-barrel core and the helical shell correspond closely to cores of stability. Surprisingly, we have also discovered a class of side chains-main chain hydrogen bonds can make very significant contributions to stability. Topology and sequence are both crucial to the folding and stability of these globular proteins.

        Tryptophan aporepressor (TR) (Figure 2) is a highly helical dimeric DNA-binding protein. The folding kinetics of TR are much more complex, involving three parallel pathways, with multiple steps along each pathway. A dimeric fragment corresponding to the first three helices that contains the hydrophobic core and the dimerization domain of the protein has been constructed and characterized. The folding kinetics of the TR core are much simpler than the full-length protein, permitting a direct measurement of the second-order rate constant for dimerization. We have currently extended our studies of oligomeric proteins to the folding of the trimeric, carbonic anhydrase-g, which contains a novel b-helix structure.

        CheY –like proteins
        CheY Protein

        Chey Protein

        Another class ofβα-repeat proteins is the CheY-like protein family, consisting of small (~ 125 residues) globular proteins that function as response regulators in bacterial two-component signal transduction pathways. Their five repeating βα units are arranged in an α/β/α sandwich topology: a central 5‑stranded β-sheet with helices 2, 3 and 4 on one surface of the sheet and helices 1 and 5 on the opposing surface. Several members of this class, including CheY, NT-NtrC and Spo0F (shown on the left), have been shown to fold via intermediates that are kinetically off the productive folding pathway. While the overall mechanism of folding for these proteins is similar and modulated by their common topology, we hypothesize that the structures of the folding intermediates and the details of the folding free-energy landscape are modulated by the amino acid sequence of the individual proteins. The sequence-local contacts between α-helices 2, 3 and 4 and the central β-sheet, define a large cluster of isoleucine, leucine and valine side chains in this early intermediate and provide a mechanism for the microsecond collapse to a kinetically-trapped species during refolding.

        Protein folding and ALS

        Protein Folding

        Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons in the brain and spinal cord. Generally ALS occurs later in life – the age of onset is generally between 40-60 years. After diagnosis, degeneration is rapid and the majority of patients die within 3 years. The largest subset of familial ALS (fALS) cases has been linked to mutations at the Cu, Zn superoxide dismutase (SOD1) locus. To date, over 150 mutations have been discovered, covering nearly 50% of the sequence and nearly all elements of secondary structure. ALS-variants of SOD1 often lead to the formation of insoluble aggregates in the motor neurons of afflicted individuals. Nevertheless, the conformations of SOD1 responsible for aggregation are unclear and a common molecular explanation for the formation of these aggregates has not been discovered. Recognizing that replacements of amino acids generally destabilize the protein, our lab seeks to quantitatively resolve the differences in the structure and folding properties of ALS-variants of SOD1 in an effort to determine a working model for aggregation and potential toxicity. It is also our intention to use insights into the folding mechanism of SOD1 to design assays for small molecular inhibitors of aggregation.


        Our lab utilizes a combination of spectroscopy and molecular biology to probe the folding properties of our various model systems. Rather than seek model systems amenable to a specific technique, our approach uses a broad array of techniques to obtain the maximum amount of information, as well as to ensure the accuracy and general applicability of the results. Furthermore, not all techniques are applicable to all proteins, due to limitations of size, solubility and stability. The broad expertise available in the lab allows for the study of systems otherwise intractable with an individual technique.

        Probes of global structure, such as circular dichroism and small angle x-ray scattering, are utilized to measure the extent of collapse, and the shape of various folded and unfolded states. On the other hand, fluorescence spectroscopy, both intensity and lifetime measurements, are used to probe the local structure of tryptophan or other extrinsic fluorescent molecules. In combination, these methods are employed to measure the stability of the fold, as well as to ascertain whether particular regions of the protein are participating in folding intermediates. More detailed information on local and global collapse reactions are obtained by utilizing Förster resonance energy transfer (FRET) techniques. These methods, which leverage the exquisite distance sensitivity of field interactions between two fluorescent molecules, allow the measurement of distances between two positions in a protein molecule during refolding and unfolding.

        To probe the extent of secondary structure formation in the native and intermediate states along a folding pathway we employ hydrogen exchange techniques coupled with either NMR or mass spectrometry. These methods exploit the fact that amide protons engaged in secondary structure formation, either by the formation of hydrogen bonds or by burial in the core of the protein, are less accessible to exchange with protons from the solvent. These techniques also hold the intriguing possibility of determining the regions of SOD1 which are involved in aggregation, by measuring the differences in protection patterns observed for the soluble protein compared to the aggregated protein.

        Rotation Projects

        Potential Rotation Projects

        1. One of the five most common structural classes of proteins is the (b/a)8 barrel motif. These proteins have eight parallel b strands arranged in a cylindrical fashion in the hydrophobic core and a corresponding number of amphipathic a-helices that dock on the surface of this cylinder. Equilibrium and kinetic studies of the folding reaction of one representative of this class, the subunit of tryptophan synthase, have shown that this single, symmetrical domain is formed by the progressive development of structure and stability in two partially folded forms. We are currently exploring the folding mechanisms of other (b/a)8 barrel proteins. Our goal for comparing the folding mechanisms of different members of the (b/a)8 barrel family is to learn how sequence relates to folding mechanism. The laboratory rotation would involve studying the equilibrium folding properties of another member of this structural class. Experiences that would be gained include expertise in protein expression and purification, and the collection and analysis of data acquired by spectroscopic techniques such as circular dichroism and fluorescence.

        2. Understanding the mechanism of folding and assembly of oligomeric peptides and proteins will provide important insights into a process that is essential for all forms of life. One model system that we have been using to study multimeric folding events is the homodimeric leucine zipper peptide, GCN4-p1. Mutational studies have yielded insights into the roles of helix propensity, salt bridges and buried polar residues in the transition state for folding. We are currently extending these studies to explore the folding and assembly of heterodimeric and heterotrimeric coiled coils. This rotation will provide training in peptide synthesis, HPLC purification, ultracentrifugation, circular dichroism spectroscopy, fluorescence resonance energy transfer experiments, and data analysis.

        3. The mechanism by which natural proteins fold to their functional forms has been evolutionarily selected to be relevant on a biological time scale.  Synthetic proteins, while designed  to achieve stable native forms, have not had evolutionary pressures applied to remove kinetic traps in their folding mechanisms and may have complicated access routes to their native states.  By studying the folding of five synthetic ├ča-repeat proteins, we will test the proposition that the design principles not only achieve the native state but do so via relatively smooth energy landscapes (Baker lab, Nature 2013).  The project will involve the expression and purification of the synthetic proteins and their biophysical characterization, with a focus on the early folding events



        selected publications
        List All   |   Timeline
        1. Broering TJ, Wang H, Boatright NK, Wang Y, Baptista K, Shayan G, Garrity KA, Kayatekin C, Bosco DA, Matthews CR, Ambrosino DM, Xu Z, Babcock GJ. Identification of Human Monoclonal Antibodies Specific for Human SOD1 Recognizing Distinct Epitopes and Forms of SOD1. PLoS One. 2013; 8(4):e61210.
          View in: PubMed
        2. Gangadhara BN, Laine JM, Kathuria SV, Massi F, Matthews CR. Clusters of Branched Aliphatic Side Chains Serve As Cores of Stability in the Native State of the HisF TIM Barrel Protein. J Mol Biol. 2013 Mar 25; 425(6):1065-81.
          View in: PubMed
        3. Kayatekin C, Cohen NR, Matthews CR. Enthalpic barriers dominate the folding and unfolding of the human cu, zn superoxide dismutase monomer. J Mol Biol. 2012 Dec 7; 424(3-4):192-202.
          View in: PubMed
        4. Kathuria SV, Guo L, Graceffa R, Barrea R, Nobrega RP, Matthews CR, Irving TC, Bilsel O. Minireview: Structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering. Biopolymers. 2011 Aug; 95(8):550-8.
          View in: PubMed
        5. Svensson AK, Bilsel O, Kayatekin C, Adefusika JA, Zitzewitz JA, Matthews CR. Metal-free ALS variants of dimeric human Cu,Zn-superoxide dismutase have enhanced populations of monomeric species. PLoS One. 2010; 5(4):e10064.
          View in: PubMed
        6. Kayatekin C, Zitzewitz JA, Matthews CR. Disulfide-reduced ALS variants of Cu, Zn superoxide dismutase exhibit increased populations of unfolded species. J Mol Biol. 2010 Apr 30; 398(2):320-31.
          View in: PubMed
        7. Yang X, Kathuria SV, Vadrevu R, Matthews CR. Betaalpha-hairpin clamps brace betaalphabeta modules and can make substantive contributions to the stability of TIM barrel proteins. PLoS One. 2009; 4(9):e7179.
          View in: PubMed
        8. Tiwari A, Liba A, Sohn SH, Seetharaman SV, Bilsel O, Matthews CR, Hart PJ, Valentine JS, Hayward LJ. Metal deficiency increases aberrant hydrophobicity of mutant superoxide dismutases that cause amyotrophic lateral sclerosis. J Biol Chem. 2009 Oct 2; 284(40):27746-58.
          View in: PubMed
        9. Noel AF, Bilsel O, Kundu A, Wu Y, Zitzewitz JA, Matthews CR. The folding free-energy surface of HIV-1 protease: insights into the thermodynamic basis for resistance to inhibitors. J Mol Biol. 2009 Apr 10; 387(4):1002-16.
          View in: PubMed
        10. Kayatekin C, Zitzewitz JA, Matthews CR. Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase. J Mol Biol. 2008 Dec 12; 384(2):540-55.
          View in: PubMed
        11. Wu Y, Kondrashkina E, Kayatekin C, Matthews CR, Bilsel O. Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein. Proc Natl Acad Sci U S A. 2008 Sep 9; 105(36):13367-72.
          View in: PubMed
        12. Kathuria SV, Day IJ, Wallace LA, Matthews CR. Kinetic traps in the folding of beta alpha-repeat proteins: CheY initially misfolds before accessing the native conformation. J Mol Biol. 2008 Oct 3; 382(2):467-84.
          View in: PubMed
        13. Vadrevu R, Wu Y, Matthews CR. NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein. J Mol Biol. 2008 Mar 14; 377(1):294-306.
          View in: PubMed
        14. Gu Z, Rao MK, Forsyth WR, Finke JM, Matthews CR. Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Go model simulation. J Mol Biol. 2007 Nov 23; 374(2):528-46.
          View in: PubMed
        15. Yang X, Vadrevu R, Wu Y, Matthews CR. Long-range side-chain-main-chain interactions play crucial roles in stabilizing the (betaalpha)8 barrel motif of the alpha subunit of tryptophan synthase. Protein Sci. 2007 Jul; 16(7):1398-409.
          View in: PubMed
        16. Forsyth WR, Bilsel O, Gu Z, Matthews CR. Topology and sequence in the folding of a TIM barrel protein: global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (betaalpha)8 barrel of unknown function from B. subtilis. J Mol Biol. 2007 Sep 7; 372(1):236-53.
          View in: PubMed
        17. Gu Z, Zitzewitz JA, Matthews CR. Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus. J Mol Biol. 2007 Apr 27; 368(2):582-94.
          View in: PubMed
        18. Wu Y, Vadrevu R, Kathuria S, Yang X, Matthews CR. A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein. J Mol Biol. 2007 Mar 9; 366(5):1624-38.
          View in: PubMed
        19. Svensson AK, Bilsel O, Kondrashkina E, Zitzewitz JA, Matthews CR. Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase. J Mol Biol. 2006 Dec 15; 364(5):1084-102.
          View in: PubMed
        20. Simler BR, Levy Y, Onuchic JN, Matthews CR. The folding energy landscape of the dimerization domain of Escherichia coli Trp repressor: a joint experimental and theoretical investigation. J Mol Biol. 2006 Oct 13; 363(1):262-78.
          View in: PubMed
        21. Svensson AK, Zitzewitz JA, Matthews CR, Smith VF. The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli. Protein Eng Des Sel. 2006 Apr; 19(4):175-85.
          View in: PubMed
        22. Bilsel O, Matthews CR. Molecular dimensions and their distributions in early folding intermediates. Curr Opin Struct Biol. 2006 Feb; 16(1):86-93.
          View in: PubMed
        23. Wu Y, Vadrevu R, Yang X, Matthews CR. Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein. J Mol Biol. 2005 Aug 19; 351(3):445-52.
          View in: PubMed
        24. Simler BR, Doyle BL, Matthews CR. Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila. Protein Eng Des Sel. 2004 Mar; 17(3):285-91.
          View in: PubMed
        25. Ibarra-Molero B, Zitzewitz JA, Matthews CR. Salt-bridges can stabilize but do not accelerate the folding of the homodimeric coiled-coil peptide GCN4-p1. J Mol Biol. 2004 Mar 5; 336(5):989-96.
          View in: PubMed
        26. Wu Y, Matthews CR. Proline replacements and the simplification of the complex, parallel channel folding mechanism for the alpha subunit of Trp synthase, a TIM barrel protein. J Mol Biol. 2003 Jul 25; 330(5):1131-44.
          View in: PubMed
        27. Vadrevu R, Falzone CJ, Matthews CR. Partial NMR assignments and secondary structure mapping of the isolated alpha subunit of Escherichia coli tryptophan synthase, a 29-kD TIM barrel protein. Protein Sci. 2003 Jan; 12(1):185-91.
          View in: PubMed
        28. Wallace LA, Matthews CR. Sequential vs. parallel protein-folding mechanisms: experimental tests for complex folding reactions. Biophys Chem. 2002 Dec 10; 101-102:113-31.
          View in: PubMed
        29. Forsyth WR, Matthews CR. Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels. J Mol Biol. 2002 Jul 26; 320(5):1119-33.
          View in: PubMed
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