Thermodynamics of a beta-hairpin structure: Evidence for cooperative formation of folding nucleus

To elucidate early nucleation stages in protein folding, multi-probed therm odynamic characterization was applied to the beta-hairpin structural format ion of G-peptide, which is a C-terminal fragment of the B1 domain of strept ococcal protein G. The segment corresponding to the sequence of G-peptide i s believed to act as a nucleus during the folding process of the B1 domain. Ln spite of the broad thermal transition of G-peptide, nuclear magnetic re sonance (NMR) melting measurements combined with our original analytical th eory enabled us to obtain the thermodynamic properties of the beta-hairpin formation with considerable accuracy. Additionally, all the thermodynamic p roperties determined by every NMR probe on both the main-chain and the side -chains were quite similar, and also comparable to the values that were ind ependently determined by calorimetric analysis of G-peptide. These results demonstrate that G-peptide folds cooperatively throughout the molecule. in other words, the formation of the beta-hairpin is interpreted as the fashio n of a first-order phase transition between two states without any distingu ishable intermediates. This cooperative formation of the short linear pepti de consisting of only 16 residues provides insight into not only the first folding events of the B1 domain, but also the general principles of protein s in terms of structural hierarchy, stability and folding mechanism. (C) 20 00 Academic Press.