Thermodynamic analysis of alpha-spectrin SH3 and two of its circular permutants with different loop lengths: Discerning the reasons for rapid foldingin proteins

The temperature dependences of the unfolding-refolding reaction of a shorte r version of the alpha-spectrin SH3 domain (PWT) used as a reference and of two circular permutants (with different poly Gly loop lengths at the newly created fused loop) have been measured by differential scanning microcalor imetry and stopped-flow kinetics, to characterize the thermodynamic nature of the transition and native states. Differential scanning calorimetry resu lts show that all these species do not belong to the same temperature depen dency of heat effect. The family of the N47-D48s circular permutant (with 0 -6 Gly inserted at the fused-loop) shows a higher enthalpy as happens with the PWT domain. The wild type (WT) and the S19-P20s permutant family have a more similar behavior although the second is far less stable. The crystall ographic structure of the PWT shows a hairpin formation in the region corre sponding to the unstructured N-terminus tail of the WT, explaining the enth alpic difference. There is a very good correlation between the calorimetric changes and the structural differences between the WT, PWT, and two circul ar permutants that suggests that their unfolded state cannot be too differe nt. Elongation of the fused loop in the two permutants, taking as a referen ce the protein with one inserted Gly, results in a small Gibbs energy chang e of entropic origin as theoretically expected. Eyring plots of the unfoldi ng and refolding semireactions show different behaviors for PWT, S19-P20s, and N47-D48s in agreement with previous studies indicating that they have d ifferent transition states. The SH3 transition state is relatively close to the native state with regard to changes in heat capacity and entropy, indi cating a high degree of compactness and order. Regarding the differences in thermodynamic parameters, it seems that rapid folding could be achieved in proteins by decreasing the entropic barrier.