Conformational unfolding in the N-terminal region of ribonuclease A detected by nonradiative energy transfer: distribution of interresidue distances in the native, denatured, and reduced-denatured states.

Abstract

AbstractTime‐dependent fluorescence measurements have been used to determine the distribution of distances between probes attached to residues 1 and (49 + 53) of bovine pancreatic ribonuclease A in the native, denatured, and reduced‐denatured states. Measurements were made on donor and on doubly labeled (donor + acceptor) protein in 50% aqueous glycerol solutions at −30°C and at room temperature. The fluorescence‐decay curves were used to compute distribution functions for the interprobe distances. The native protein has a narrow distribution of interprobe distances at −30°C (high‐viscosity medium); this distribution is narrower at room temperature (low‐viscosity medium), due primarily to the dynamic flexibility of the probes. Denaturation by 6M guanidine hydrochloride leads to a wider distribution of distances at −30°C, with a shift of the distribution curve to larger distances, because of the increased disorder of the protein. Reduction of the disulfide bonds by dithiothreitol leads to further decreases in transfer efficiency (a unique distribution curve for the reduced protein was not obtained because of the low transfer efficiency). Both the denatured and reduced‐denatured species have average interprobe distances of about 60 Å, compared to 36 Å for the native protein. Reduction of the solvent viscosity leads to nearly monoexponential decay of the donor fluorescence in the doubly labeled derivative. This is interpreted as a manifestation of fast local Brownian motions. It appears that large‐scale segmental motions do not take place in the denatured protein within the excited‐state lifetime of the donor (ca. 8 ns). The above results indicate that reduced‐denatured ribonuclease A has residual structure that limits segmental Brownian motion in the N‐terminal segment.