Exploring the role of the unfolded state in protein folding

We analyzed the energetic and structural role of the unfolded state in the folding of gsmFKBP protein. Three mutants of gsmFKBP were generated based on information about existing residual structure of the protein in the urea-unfolded state. Mutations replaced the residue Q53 with better N-capping residue: N, T and D, in order to enhance the helix formation in the unfolded state. We proposed that mutations would change the residual structure and energy of the urea-unfolded state, which would effect kinetic and thermodynamic properties of the mutated proteins.;We found that mutations have small effect on overall protein stability. The gsmFKBP and its Q53N, Q53T and Q53D mutants follow two-state equilibrium isothermal urea-induced denaturation transition.;The kinetic data on rates of folding and unfolding suggest that two energy states are changed in mutated proteins, unfolded and transition. We found that gsmFKBP and Q53N protein fold via kinetic intermediate, whereas Q53T and Q53D proteins do not have any folding intermediate states. Mutations to T or D abolished the intermediate formation and altered protein folding pathway. Mutations in position Q53 of gsmFKBP resulted in large, nonclassical phi-values, which indicate that this residue serves as a "gatekeeper" defining the protein folding pathway.;We showed by NMR that the native structures of gsmFKBP and its mutants are very similar. However, measurable differences in nonrandom secondary structure of gsmFKBP and its mutants were identified in the urea-unfolded state. Observed differences in NOES patterns suggest that each mutant has enhanced helix formation in urea-unfolded state compared to gsmFKBP.;Our results provide first experimental evidence that nonrandom secondary structure in the unfolded state can change a protein folding pathway.