The Kinetic and Equilibrium Molten Globule Intermediates of Apoleghemoglobin Differ in Structure

Abstract

An important question in protein folding is whether molten globule states formed under equilibrium conditions are good structural models for kinetic folding intermediates. The structures of the kinetic and equilibrium intermediates in the folding of the plant globin apoleghemoglobin have been compared at high resolution by quench-flow pH-pulse labeling and interrupted H/D exchange analyzed in DMSO solvent. Unlike its well-studied homolog, apomyoglobin, where the equilibrium and kinetic intermediates are quite similar, there are striking structural differences between the intermediates formed by apoleghemoglobin. In the kinetic intermediate, formed during the burst phase of the quench flow experiment, protected amides and helical structure are found mainly in the regions corresponding to the G and H helices of the folded protein, and in parts of the E helix and CE loop regions, whereas in the equilibrium intermediate, amide protection and helical structure are seen in parts of the A and B helix regions, as well as in the G and H regions, and the E helix remains largely unfolded. These results suggest that the structure of the molten globule intermediate of apoleghemoglobin is more plastic than that of apomyoglobin, so that it is readily transformed depending on the solution conditions, particularly pH. Thus, in the case of apoleghemoglobin at least, the equilibrium molten globule formed under destabilizing conditions at acid pH is not a good model for the compact intermediate formed during kinetic refolding experiments. Our high-precision kinetic analysis also reveals an additional slow phase during the folding of apoleghemoglobin, which is not observed for apomyoglobin. Hydrogen exchange pulse labeling experiments show that the slow folding phase is associated with residues in the C-E loop, which probably forms non-native structure in the intermediate that must be resolved before folding can proceed to completion.

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

“…With the exception of Tyr 114 , these protection factors are low and do not exceed a value of ϳ300 (Table 1). These protection factors are comparable with those observed for the molten globules of apomyoglobin (36), apoleghemoglobin (41), and RNase HI (42). In contrast, backbone amides of native apoflavodoxin have protection factors up to 2 ϫ 10 6 .…”

“…This ordering is due to ␣-helix formation and local and non-local hydrophobic interactions. The ordered regions comprise residues Ala 41 (44). These structured elements in the unfolded protein transiently interact and subsequently form the ordered core of the off-pathway molten globule.…”

Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule

“…Extensive experiments on apomyoglobin have shown that the hydrogen-exchange protection in the acid state molten globule follows a slightly different pattern and has a different stability distribution than observed in the kinetic intermediate (26,27). In a related protein, apoleghemoglobin, there are substantial structural differences between the protection in the acid state molten globule and the kinetic intermediate (28). Though conformational heterogeneity has been observed in the kinetic intermediate of apomyoglobin, such observations are not incompatible with a region of structure that remains well folded (25,27,29).…”

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

“…Structural characterization of intermediates or MG state that populate in the folding/unfolding process is essential to understand the protein folding mechanism [4]. At MG state, the protein retains most of the secondary structures, but has a tendency to lose some of the tertiary structures [5][6][7]. The MG states are believed to be general folding intermediates because they populate both in the equilibrium and kinetic folding/ unfolding pathways [8].…”