Properties and crystal structure of a beta-barrel folding mutant

A mutant of a beta-barrel protein, rat intestinal fatty acid binding protei n, was predicted to be more stable than the wild-type protein due to a nove l hydrogen bond. Equilibrium denaturation! studies indicated the opposite: the V60N mutant protein was less stable. The folding transitions followed b y CD and fluorescence were reversible and two-stale for both mutant and wil d-type protein. However, the rates of denaturation,and renaturation of V60N were faster. During unfolding, the initial-rate was associated with 75-80% of the fluorescence and all of the CD amplitude change, A subsequent rate accounted for the remaining fluorescence change for both proteins; thus: th e intermediate state lacked secondary structure. During folding, one rate w as detected by both fluorescence and CD after an initial burst phase for bo th wild-type and mutant. An additional slower folding rate was detected by fluorescence for the mutant:protein. The structure of the V60N mutant has b een obtained and is nearly identical to prior crystal structures of IFABP. Analysis of mean differences in hydrogen bond and van der Waals interaction s did not readily account for the stability loss due to the mutation. Howev er, significant average differences of the solvent accessible surface and c rystallographic displacement factors suggest entropic destabilization.