Structural and kinetic characterization of early folding events in β-lactoglobulin

We have defined the structural and dynamic properties of an early folding intermediate of beta -lactoglobulin known to contain non-native alpha -helical structure. The folding of beta -lactoglobulin was monitored over the 100 mus-10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR An initial increase in Trp fluorescence with a time constant of 140 mus is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta -strands F, G and ii, and the main alpha -helix, which was thus identified as the folding core of beta -lactoglobulin, At the same time, weak protection (up to similar to 10-fold) of amide protons in a segment spanning residues 12-21 is consistent with formation of marginally stable nonnative alpha -helices near the N-terminus, Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.