AMIDE HYDROGEN-EXCHANGE AND INTERNAL DYNAMICS THE CHEMOTACTIC PROTEINCHEY FROM ESCHERICHIA-COLI

The backbone internal dynamics of the wild-type 129 amino acid alpha/b eta parallel protein CheY and its double mutant F14N/P110G are analyse d here by the hydrogen-exchange method. The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is desta bilising and accelerates unfolding. We first assigned and characterise d the double mutant by nuclear magnetic resonance (NMR), to try and di scover any-possible conformational change induced by the two mutations . The main difference between the two proteins is a favourable N-cappi ng interaction of the newly introduced Asn14 side-chain at the beginni ng of the first alpha-helix (alpha-helix A). Second, we have measured the exchange rates in the wild-type and mutant CheY. In the first case the observed protection factors are slightly dispersed around an aver age value. According to their distribution in the structure, protein s tability is highest on one face of the central beta-sheet, in the surr oundings of the main hydrophobic core formed by side-chains of residue s in beta-strands I, II and III and helices A and E. The mutations in the double mutant protein affect two distinct subdomains differently ( from beta-strand I to III and from alpha-helix C to the end). In the s econd subdomain the number of protected protons is reduced with respec t to those in the wild-type. This differential behaviour can be explai ned by a selective decrease in stability of the second folding subdoma in produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation. alpha-Helix A, which is inv olved together with beta-strands I and III in the folding nucleus of C heY, shows the largest protection factors in both proteins. (C) 1997 A cademic Press Limited.