Heat-resistant structural features of bovine β-lactoglobulin A revealed by NMR H/D exchange observations

The thermal unfolding of the A variant of bovine beta-lactoglobulin (beta-Lg), in 50 mm phosphate buffer at pH 3.0, was followed by nuclear magnetic resonance (NMR) hydrogen/deuterium (H/D) exchange observations. The exchange behaviour of a large number of backbone amide protons (HNS) was monitored at temperatures between 37degreesC and 80degreesC to determine the relative thermal stability of different elements of the native protein structure. The H/D exchange was rapid at 37degreesC for the HNS on the residues in loops and in the terminal regions of the native protein. Further H/D exchange was promoted by heat treatment at temperatures up to 80degreesC and was completed for the identifiable H-N of the various alpha and beta structural elements in the following order: D-E strand (55-60degreesC); C-D strand and a-helix (60-65degreesC); A-B, A-I and E-F strands (65-70degreesC); and A-H, B-C and F-G strands (75-80degreesC). At WC, the only identifiable H-N signal was from F105 which indicated that the G-H pair of disulphide-linked strands formed the most heat-resistant feature of the beta-Lg structure. The effect of heating to 80degreesC was shown to be largely reversible by subsequent D/H exchange and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Interestingly, the susceptibility of particular HNS to H/D exchange was found to correlate reasonably well with the susceptibility to tryptic hydrolysis. (C) 2002 Elsevier Science Ltd. All rights reserved.