Effects of core mutations on the folding of a beta-sheet protein: Implications for backbone organization in the I-State

A series of core mutations were introduced into beta-strand segments of an immunoglobulin fold (the isolated first domain of CD2, CD2.d1) to examine t heir influence on the rapidly formed intermediate state (I-state) which tra nsiently accumulates in the folding reaction [Parker, M. J., and Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. The residue changes were chemically conservative, each representing the removal of one or two methylene groups from aliphatic side chains. Predictably, the mutations destabilize the fol ded state with respect to the unfolded state by about 1.1 +/- 0.7 kcal mol( -1) per methylene group removed. However, when the folding reaction is diss ected by transient kinetic analysis into its component steps, six out of th e nine mutations lead to a stabilization of the I-state. The direction and magnitude of these effects on the global stability of the transient interme diate are well correlated with changes in secondary structure propensity oc casioned by the substitutions. The results show that, although side chain i nteractions are extremely weak in this early phase of folding, the beta-str and conformation of the polypeptide chain is established. In the next phase of the reaction, the rate-limiting transition state is attained by the for mation of a tightly localized hydrophobic nucleus which includes residues V 30, I18, and V78. Interestingly, in almost all immunoglobulin domains of ex tracellular proteins, the latter pair are cysteine residues which form a di sulfide bridge.