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

Abstract

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. thus form the stable core of the helical molten globule of ␣-␤ parallel flavodoxin, which is almost entirely structured. Nonnative docking of helices in the molten globule of flavodoxin prevents formation of the parallel ␤-sheet of native flavodoxin. Hence, to produce native ␣-␤ parallel protein molecules, the off-pathway species needs to unfold.Non-native protein conformations initially drew attention by their importance for the understanding of the process of protein folding (1-3). Now it is recognized that these conformations also yield important information about protein misfolding and aggregation (4). Partially folded states of proteins with exposed hydrophobic surfaces, such as molten globules, are prone to aggregation and can be precursors of amyloid fibril formation. This aggregation phenomenon can have devastating effects on organisms (4). The resemblance between early kinetic intermediates and molten globules (5-8) suggests that these molten globules can be considered as models of transient intermediates (9). This resemblance has been demonstrated for ␣-lactalbumin (10 -12), apomyoglobin (13,14), RNase H (15), T4 lysozyme (16), Im7 (17), and flavodoxin (18). Understanding the formation and conformation of these molten globules offers insights into factors responsible for protein misfolding and, potentially, for numerous debilitating pathologies (19).Upon descending the folding funnel, proteins encounter folding energy landscapes that are rough (20, 21). As a result, partially folded intermediates, which may be on-or off-pathway to the native state, are populated. When the intermediate is on-pathway, as is observed for the majority of proteins studied to date, it has a native-like topology and is productive for folding. In contrast, when the intermediate is off-pathway, it is trapped in such a manner that the native state cannot be reached without substantial reorganizational events (9). Several kinetic studies have revealed involvement of off-pathway intermediates during protein folding (18,22,23). A decrease of the folding rate due to the presence of an off-pathway molten globule, which is kinetically trapped and partially folded, increases the likelihood of protein aggregation.Here, using H/D exchange 2 experiments, we report the characterization of the off-pathway molten globule folding intermediate of a 179-residue flavodoxin from Azotobacter vinelandii. Flavodoxins are monomeric proteins involved in electron transport and contain a non-covalently bound FMN cofactor. The proteins consist of a single structural domain and adopt t...

The ribosome regulates flavodoxin folding

“…In addition, the ribosome seems to have a protein folding activity through Domain V rRNA (70). This folding activity has been shown to promote productive folding from denatured and MG states (71)(72)(73). As both MGs of F44Y apoflavodoxin arise under physiological conditions, these MGs are promising candidates for the investigation of how a cell deals with their potential intracellular formation.…”

“…Its off-pathway nature is due to docking of non-native α-helices, thereby preventing formation of the central β-sheet of native flavodoxin (Fig. 4) (70, 71,78). Formation of off-pathway folding intermediates seems to be typical for proteins with a flavodoxin-like fold, as they have also been observed for Anabaena apoflavodoxin and chemotactic protein (59,(79)(80)(81).…”

“…MG off aggregates severely at elevated protein concentrations or in the presence of macromolecular crowders (77). While formation of native apoflavodoxin is highly cooperative, MG off folds non-cooperatively (71). In vitro, the folding of the C-terminal part of MG off precedes the folding of the N-terminal part and the MG gradually compacts due to progressive extension of its ordered core (74).…”

“…Due to its instability it is not observed during denaturant-dependent equilibrium folding. In contrast, the off-pathway intermediate MG off is a molten globule that is relatively stable and needs to unfold extensively before productive folding to the native state can resume (71,77). Of all folding apoflavodoxin molecules approximately 90 % first misfolds to this MG (67).…”

“…The folding of flavodoxin II from A. vinelandii (65,66) has been studied extensively. Un-and re-folding of both the apo-and the holo-form of this protein have been probed, using GuHCl as denaturant (67)(68)(69)(70)(71)(72)(73)(74). It has been shown that apoflavodoxin folds autonomously to its native state, after which it binds FMN as the last step in flavodoxin formation (Fig.…”