The temperature dependences of the unfolding-refolding reaction of a shorter 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 microcalorimetry and stopped-flow kinetics, to characterize the thermodynamic nature of the transition and native states. Differential scanning calorimetry results show that all these species do not belong to the same temperature dependency 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 crystallographic structure of the PWT shows a hairpin formation in the region corresponding to the unstructured N-terminus tail of the WT, explaining the enthalpic difference. There is a very good correlation between the calorimetric changes and the structural differences between the WT, PWT, and two circular permutants that suggests that their unfolded state cannot be too different. Elongation of the fused loop in the two permutants, taking as a reference the protein with one inserted Gly, results in a small Gibbs energy change of entropic origin as theoretically expected. Eyring plots of the unfolding and refolding semireactions show different behaviors for PWT, S19-P20s, and N47-D48s in agreement with previous studies indicating that they have different transition states. The SH3 transition state is relatively close to the native state with regard to changes in heat capacity and entropy, indicating 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.

• PMID: 9888794
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Martínez JC, Viguera AR, Berisio R, Wilmanns M, Mateo PL, Filimonov VV, Serrano L

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