ENGINEERED TYROSINE RESIDUES SERVE AS THE LOCAL PROBES TO DETECT A KINETIC INTERMEDIATE IN THE FOLDING OF RIBOSE-BINDING PROTEIN

Ribose-binding protein (RBP) consists of two α/β globular units; the N domain being composed of four helices (I, II, III and IX) and six sheets (A, B, C, D, E and K), and the C domain with five helices (IV, V, VI, VII and VIII) and six sheets (P, G, H, I, J and L). The two domains are connected by three strands. In the previous study, tyrosine residues in the RBP were substituted by phenylalanine to examine the fluorescence property of each chromophore. Since the three tyrosine residues are scattered in the two domains of RBP, residues 32 and 261 in the N domain and 115 in the C domain, we were able to monitor the state of protein folding using unaltered tyrosine as a local probe. The final structures of the mutant proteins show little differences from those of wild-type as judged by circular dichroism spectra. The equilibrium and kinetic folding behaviors of the mutant RBPs were examined by fluorescence spectroscopy. The equilibrium data obtained from the mutant RBPs conform to the two-state transition involving the native and unfolded species. However, kinetic studies indicate that there exists an intermediate formed transiently during the folding or unfolding process, which was not detected in equilibrium experiments. In unfolding kinetics of the mutant protein retaining only the N domain tyrosine residues, a striking change in fluorescence was observed as an initial jump, suggesting the presence of a partial unfolding step of RBP around the Tyr32 chromophore, since the fluorescence of Tyr261 does not change upon folding. This occurred immediately after mixing with 0.6 to I M range of guanidine hydrochloride and was completed in less than three seconds of the mixing dead time. The partial increase of fluorescence appears to be due to the dissociation of a quenching group, the carboxyl side-chain of Asp249 located on helix IX, from the Tyr32 fluorophore. The successive unfolding process reflects a process of release from the second quenching group at Asp2 on the sheet A. The substitution at Tyr261 on sheet K by phenylalanine reveals an additional kinetic phase in refolding, in which the folding from unfolded into an intermediate form seems similar to wild-type in time scale, whereas the next step leading to the formation of native protein becomes slower. The removal of hydrogen bonds between Tyr261 and Glu246 in the nearby helix IX, due to the introduction of phenylalanine, appears to cause an increase in the activation free energy for the dissociation of Asp249, on helix IX, from Tyr32 of sheet B during unfolding. This implies that an association between sheet B and helix IT; plays a key role in maintaining the kinetic intermediate in the same manner as indicated by the study of N domain folding just described. Based on the structural information that the completion of N domain conformation involves the addition of a helix IX-loop-sheet K structural unit, extended from the C domain, to the remainder of a pre-N domain complex, it is proposed that this cross-bridge addition provides a unique kinetic barrier in the folding pathway of RBP. © 1994 Academic Press Limited.