C. Robert Matthews, Ph.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Biochemistry and Molecular Pharmacology

Other Affiliation(s):
   Center for AIDS Research

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

Solving the Protein Folding Problem

Determining the mechanism by which the amino acid sequence of a protein directs the rapid and efficient folding to the native, functional conformation is one of the most challenging problems in molecular biophysics. 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). Advances in genetic engineering, peptide synthesis and spectroscopy provide new insight into the structure and stability of folding intermediates, i.e., partially folded forms that contain essential clues on the mechanism.

In our laboratory we use these new technologies in a broad-ranging effort to solve the protein folding problem. By combining site-directed mutagenesis, protein fragmentation or peptide synthesis with biophysical techniques (including absorbance, fluorescence, circular dichroism and NMR spectroscopies), insights into the folding mechanisms of several representative proteins are being obtained. Current efforts are focused on the folding of several monomeric proteins, including the a-subunit of tryptophan synthase from E. coli, dihydrofolate reductases from E. coli and human, and the ras p21 protein from human. The folding of oligomeric proteins and the factors influencing the association of protein chains are also being addressed with studies on the tryptophan aporepressor from E. coli, carbonic anhydrase from M. thermophila, and a peptide model system, the leucine zipper domain of the yeast transcriptional activator, GCN4.

Figure 1

GCN4-pl (Figure 1) is a 33 amino acid peptide that forms a coiled-coil structure composed of two right-handed a-helices that wrap around each other to form a left-handed supertwist. The simple two-state folding kinetics of GCN4-pl show no evidence of detectable intermediates. Mutational and fragmentation studies on GCN4-p1 suggest that specific structure is required in the monomer chains for productive dimerization.

Figure 2

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.

Figure 3

The a subunit of tryptophan synthase (Figure 3) has an a/b barrel structure, a motif found in more than two dozen other proteins. 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 have shown that this single, symmetrical domain is formed by the progressive development of structure and stability in two partially folded forms. Recent experiments have addressed the three-dimensional structures of these two intermediates; the structures, stabilities and folding kinetics of independently folding fragments of the protein; and the relationships between the stable intermediates and these independently folding substructures.

Figure 4

Dihydrofolate reductase (DHFR) (Figure 4) is a single polypeptide chain that folds into an a/b sheet motif containing two structural domains. Stopped-flow circular dichroism and fluorescence spectroscopy as well as quench-flow NMR spectroscopy on the E. coli protein show that specific, native-like secondary structure and nonpolar surfaces form within 5 milliseconds. Further folding occurs through four parallel folding channels to a set of native conformers via rate-limiting folding reactions. The folding of the human DHFR variant and the ras p21 protein show similar parallel folding events. This finding suggests that their folding mechanisms reflect the global properties of the motif, and not the specific properties of the amino acid sequences.

The goal of all of these studies is to understand the principles of how the sequence of a protein determines its three-dimensional structure. The development of a folding code would greatly enhance the information content of the DNA sequences that have been generated by the Human Genome Project. Other benefits of such a code would be an enhanced understanding of disease states caused by misfolded proteins and an improved ability to design proteins and enzymes de novo for creation of new synthetic or therapeutic agents.

Representative Publications

Wu,Y., Matthews, C.R. "Proline replacements and the simplification of the folding mechanism for the alpha subunit of Trp synthase, a TIM barrel protein." J. Mol. Biol., 330, 1131-1144 (2003).

Aria, M., Kataoka, M., Kumajima, K., Matthews, C.R., Iwakura, M. "Effects of the Difference in the Unfolded-state Ensemble on the Folding of Escherichia coli Dihydrofolate Reductase."    J. Mol. Biol. 329, 779-791 (2003).

Svensson, A.K., O'Neil, J.C. Jr., Matthews, C.R. "The Coordination of the Isomerization of a Connserved Non-prolyl cic Peptide Bond with the Rate-limiting Steps in the Folding of Dihydrofolate Reductase." J. Mol. Biol. 326, 569-583 (2003).

Vadrevu, R. Falzone, C.J., Matthews, C.R. "Partial NMR Assignements and Secondary Structure Mapping of the Isolated Subunits of Escherichia coli tryptophan synthase, a 29kDa TIM Barrel Protein." Protein Science, 12, 185-191 (2003).

Knappenberger, J.A., Smith, J.E., Thorpe, S.H., Zitzewitz, J.A., Matthews, C.R. A Buried Polar Residue in the Hydrophobic Interface of the Coiled-coil Peptide, GCN4-p1, Plays a Thermodynamic, not a Kinetic Role in Folding. J. Molec. Biol, 321, 1-6 (2002).

Forsyth, W.R., Matthews, C.R. Folding Mechanism of Indole-3-glycerol Phosphate Synthase from Sulfurous solfactaricus: A Test of the Conservation of Folding Mechanisms Hypothesis in (ba)8 Barrels. J. Molec. Biol., 320, 1119-1133, (2002).

Steinbach, P.J., Ionescu, R., Matthews, C.R. Analysis of Kinetics Using a Hybrid Maximum-Entropy/Nonlinear-Least-Squares Method: Application to Protein Folding. Biophys. J., 82, 2244-2255, (2002).

Wallace, L. and Matthews, C.R. Highly Divergent Dihydrofolate Reductases Conserve Complex Folding Mechanisms. J. Molec. Biol. 315, 193-211, (2002).

Gloss, L. M., Simler, B. R., Matthews, C. R. Rough Energy Landscapes in protein Folding: Dimeric E. coli Trp Repressor Folds Through Three Parallel Channels. J. Molec. Biology 312, 1121-1134, (2001).

Smith, V. F., Matthews, C. R. Testing the Role of Chain Connectivity on the Stability and Structure of Dihydrofolate Reductase from E. coli: Fragment Complementation and Circular Permutation Reveal Stable, Alternatively Folded Forms. Protein Science, 10, 116-128 (2001).

Ibarra-Molero, B., Makhatadze, G. I., Matthews, C. R. Mapping the Energy Surface for the Folding Reaction of the Coiled-Coil Peptide GCN4-p1. Biochemistry, 40, 719-731 (2001).

Bilsel, O., Matthews, C. R. (2000) Barriers in protein folding reactions. Advances in Protein Chemistry 53, 154-207.

Zitzewitz J.A., Ibarra-Molero, B., Fishel, D.R., Terry, K.L., Matthews C.R. (2000) Preformed secondary structure drives the association reaction of GCN4-p1, a model coiled system. J. Mol. Biol. 296, 1105-1116.

Ionescu, R.M., Smith, V.F., O'Neill Jr., J.C., Matthews, C.R. (2000) Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles. Biochemistry 39, 9540-9550.

O'Neill Jr, J.C., Matthews, C.R. (2000) Localized, stereochemically-sensitive hydrophobic packing in an early intermediate of dihydrofolate reductase of Escherichia coli. J. Mol. Biol. 295, 737-744.

Zitzewitz J.A., Matthews C.R. (1999) Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein. Biochemistry 38, 10205-10214.

Gualfetti, P., Iwakura, M., Lee, J., Kihara, H., Bilsel, O., Zitzewitz J.A., Matthews C.R. (1999) Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein. Biochemistry 38, 13367-13378.

Gualfetti, P., Bilsel, O., Matthews C.R. (1999) The progressive development of structure and stability during the folding of the alpha subunit of tryptophan synthase from E. coli. Protein Science 8, 1623-1635.

Bilsel O., Zitzewitz J.A., Bowers K.E., Matthews C.R. (1999) Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels. Biochemistry 38, 1018-1029.

Bilsel, O., Yang, L., Zitzewitz J.A., Beechem, J., Matthews C.R. (1999) Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals a non-monotonic behavior of the rotational correlation time. Biochemistry 38, 4177-4187.

Zitzewitz J.A., Gualfetti, P.J., Perkons, I.E., Wasta, S.A., Matthews C.R. (1999) Identifying the structural boundaries of independent folding domains in the alpha/beta barrel protein, alpha tryptophan synthase. Protein Science 8, 1200-1209.

Zhang, J., Matthews, C.R. (1998) The role of ligand binding in the kinetic folding mechanism of human p21 (H-ras) protein. Biochemistry 37, 14891-14899.

Zhang, J., Matthews, C.R. (1998) Ligand binding is the principal determinant of stability for the p21 (H-ras) protein. Biochemistry 37, 14881-14890.

Gloss, L.M., Matthews C.R. (1998) Mechanism of folding of the dimeric core domain of Escherichia coli trp repressor: a nearly diffusion-limited reaction leads to the formation of an on-pathway dimeric intermediate. Biochemistry 37, 15990-15999.

Gloss L.M., Matthews C.R. (1998) The barriers in the bimolecular and unimolecular folding reactions of the dimeric core domain of Escherichia coli Trp repressor are dominated by enthalpic contributions. Biochemistry 37, 16000-16010.

Shao X., Matthews C.R. (1998) Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate. Biochemistry 37, 7850-7858.

Goldberg, M., Zhang, J., Sondek, S., Matthews, C.R., Fox, R., Horwich, A. (1997) Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL. Proc. Natl. Acad. Sci. USA 94, 1080-1085.

Gegg C.V., Bowers K.E., Matthews C.R. (1997) Probing minimal independent folding units in dihydrofolate reductase by molecular dissection. Protein Science 6, 1885-92.

Gegg C.V., Bowers K.E., Matthews C.R. (1997) A general approach for the design and isolation of protein fragments: the molecular dissection of dihydrofolate reductase in Techniques in Protein Chemistry VII (Marshak, D., Ed.) pp. 439-448, Academic Press, Inc., San Diego, CA.

Shao X., Hensley P., Matthews C.R. (1997) Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein. Biochemistry 36, 9941-9949.

Gloss L.M., Matthews C.R. (1997) Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor.Biochemistry 36, 5612-5623.

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.

Laboratory Staff

Coming soon

Academic Background

Office: LRB 928, Lab 970 A-D
Phone: 508 856-2251
E-mail: C.Robert.Matthews@umassmed.edu
Keywords: Protein Folding, Biophysics, Biochemistry

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