Distributions of intramolecular distances in the reduced and denatured states of bovine pancreatic ribonuclease A. Folding initiation structures in the C-terminal portions of the reduced protein

The purpose of this investigation is to characterize the reduced state of R Nase A (r-RNase A) in terms of (i) intramolecular distances, (ii) the seque nce of formation of stable loops in the initial stages of folding, and (iii ) the unfolding transitions induced by GdnHCl. This is accomplished by iden tifying specific subdomain structures and local and long-range interactions that direct the folding process of this protein and lead to the native fol d and formation of the disulfide bonds. Eleven pairs of dispersed sites in the RNase A molecule were labeled with fluorescent donor and acceptor probe s, and the distributions of intramolecular distances (IDDs) were determined by means of time-resolved dynamic nonradiative excitation energy transfer (TR-FRET) measurements. The mutants were designed to search for (a) a possi ble nonrandom fold of the backbone in the collapsed state and (b) possible loops stabilized by lone-range interactions, It was found that, under foldi ng conditions, (i) the labeled mutants of r-RNase A in refolding buffer (th e RN state) exhibit features of specific (nonrandom) compact but very dispe rsed subdomain structures (indicated by short mean distances, broad IDDs, a nd a weak dependence of the mean distances on segment length), (ii) the bac kbone fold in the C-terminal beta -like portion of the molecule appears to adopt a native-like overall fold, (iii) the N-terminal alpha -like portion of the chain is separated from the C-terminal core by very large intramolec ular distances, larger than those in the crystal structure, and (iv) pertur bations by addition of GdnHCl reveal several conformational transitions in different sections of the chain. Addition of GdnHCl to the native disulfide -intact protein provided a reference state for the extent of expansion of i ntramolecular distances under denaturing conditions. In conclusion, r-RNase A under folding conditions (the RN state) is poised for the final folding step(s) with a native-like trace of the chain fold but a large separation b etween the two subdomains which is then decreased upon introduction of thre e of the four native disulfide cross-links.