| The primary amino acid sequence of a protein contains sufficient information to encode for the tertiary fold of a protein. A purely theoretical solution for the protein folding problem, however, has not yet been feasible. Therefore, the experimental characterization of intermediates formed during refolding can provide information which may contribute to a better understanding of the folding process, and provide insight into the interactions which give rise to the stability of the final folded structure.;The detection of structural kinetic folding intermediates is complicated by the finding that the unfolded state of most proteins are heterogeneous. It has been proposed that the origin of this heterogeneity is due to a mixture of cis and trans peptide bond isomers preceding proline amino acids in the unfolded polypeptide chain. Although a large number of studies have provided evidence in support of this hypothesis, particularly in regards to the folding pathway of RNase A, direct verification of the cause of the heterogeneity has been elusive.;A system has been developed which allows for the expression of RNase A in Escherichia coli and its purification. Substitution of the prolines, which are cis in the folded structure, is performed by site-directed mutagenesis. As judged by protein refolding, the heterogeneity in the unfolded state of the protein is eliminated by substitution of both cis prolines.;The effect on both the structure and stability of the folded state caused by cis-proline substitution is examined. As judged by thermal or chemical denaturation experiments, these mutations destabilize the folded structure quite dramatically. The destabilization reflects the fact that the introduced amino acids must either alter the final folded structure, if they are trans, or they must be forced into the cis conformation. Preliminary 2-D NMR studies provide evidence that in the case of P93A (proline 93 to alanine) the peptide bond preceding alanine 93 does exist in the cis conformation. |
