Robert L. Baldwin, Emeritus

    Our laboratory is focused on the mechanism of protein folding. The basic strategy is to characterize the structures of folding intermediates to learn what interactions are dominant at a given stage in folding. Experiments in protein engineering are used to test what has been learned. We have had some success with two approaches.

The first approach is to study the folding of a protein fragment, to find out how much of its structure is determined by its own amino acid sequence and how much by interactions with distant residues that interact only when the tertiary structure is formed. The C-peptide contains residues 1-13 of ribonuclease A (RNase A), including residues 3-13 which form an a-helix in intact RNase A. Although C-peptide does not form a stable helix at 25°C in H2O, partial helix formation can be measured near 0°C. In collaboration with John Stewart at the University of Colorado Medical School, we have used peptide synthesis to find helix-stabilizing substitutions and to learn which residues of C-peptide are involved in specific helix-stabilizing interactions. The results show that interactions involving charged groups (salt bridges and interactions with the a-helix dipole) play a major role in controlling helix stability of short peptides in H2O. The results also show that the structure of the helix formed by isolated C-peptide is surprisingly close to that of the helix formed by residues 3-13 in RNase A. More recent experiments with peptides of de novo design make use of the high helix propensity of alanine to study helix propensities of other amino acids in an alanine background.

The second approach is to find structural intermediates in folding by analyzing the kinetics of folding of the intact protein near 0°C. Structural intermediates in the folding of RNase A are well-populated in these conditions but folding is rapid even near 0°C. A basic tool in the analysis of kinetic intermediates is the exchange reaction of peptide NH protons with D2O or 3H-H2O, since the formation of a-helices or b-sheets drastically retards exchange and thus structure present at a given stage in folding can be "trapped" by preventing exchange of peptide NH protons. Folding intermediates have been labeled with 3H-H2O and the structural stability of the intermediates has been analyzed. In current work, high-resolution 1H-NMR is being used to determine which peptide NH protons are protected against exchange at different stages of folding, and in this way to determine the kinetic pathway of folding. Similar studies of an equilibrium "molten globule" intermediate of apomyoglobin show that some of the a-helices of the native protein are present at an early stage of folding in this intermediate.

Barrick, D. and Baldwin, R. L. (1993) Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding. Protein Sci 2, (6): 869-876. (Medline)

Chakrabartty, A., Kortemme, T. and Baldwin, R. L. (1994) Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side-chain interactions. Protein Sci. 3, 843-852. (Medline)

Huyghes-Despointes, B.M.P., Klingler, T.M. and Baldwin, R.L. (1995) Measuring the Strength of Side-Chain Hydrogen Bonds in Peptide Helices: The Gln·Asp (i,i+4) Interaction. Biochem 34, 13267-13271. (Medline)

Kay, M.S. and Baldwin R.L. (1996) Packing interactions in the apomyoglobin folding intermediate. Nature Structural Biology 3,(5): 439-445. (Medline)

Loh, S.N., Rohl, C.A., Kiefhaber, T. and Baldwin, R.L. (1996) A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions. Proc. Natl. Acad. Sci. USA 93, 1982-1987. (Medline)