Section: Research

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.

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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