Interrupted hydrogen/deuterium exchange reveals the stable core of the remarkably helical molten globule of alpha-beta parallel protein flavodoxin.

Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule that is observed during folding of alpha-beta parallel flavodoxin from Azotobacter vinelandii is a remarkably non-native species. This folding intermediate is helical and contains no beta-sheet and is kinetically off-pathway to the native state. It can be trapped under native-like conditions by substituting residue Phe(44) for Tyr(44). To characterize this species at the residue level, in this study, use is made of interrupted hydrogen/deuterium exchange detected by NMR spectroscopy. In the molten globule of flavodoxin, the helical region comprising residues Leu(110)-Val(125) is shown to be better protected against exchange than the other ordered parts of the folding intermediate. This helical region is better buried than the other helices, causing its context-dependent stabilization against unfolding. Residues Leu(110)-Val(125) thus form the stable core of the helical molten globule of alpha-beta parallel flavodoxin, which is almost entirely structured. Non-native docking of helices in the molten globule of flavodoxin prevents formation of the parallel beta-sheet of native flavodoxin. Hence, to produce native alpha-beta parallel protein molecules, the off-pathway species needs to unfold.