Extensive Formation of Off-Pathway Species during Folding of an α−β Parallel Protein Is Due to Docking of (Non)native Structure Elements in Unfolded Molecules

Abstract

Detailed information about unfolded states is required to understand how proteins fold. Knowledge about folding intermediates formed subsequently is essential to get a grip on pathological aggregation phenomena. During folding of apoflavodoxin, which adopts the widely prevalent alpha-beta parallel topology, most molecules fold via an off-pathway folding intermediate with helical properties. To better understand why this species is formed, guanidine hydrochloride-unfolded apoflavodoxin is characterized at the residue level using heteronuclear NMR spectroscopy. In 6.0 M denaturant, the protein behaves as a random coil. In contrast, at 3.4 M denaturant, secondary shifts and (1)H-(15)N relaxation rates report four transiently ordered regions in unfolded apoflavodoxin. These regions have restricted flexibility on the (sub)nanosecond time scale. Secondary shifts show that three of these regions form alpha-helices, which are populated about 10% of the time, as confirmed by far-UV CD data. One region of unfolded apoflavodoxin adopts non-native structure. Of the alpha-helices observed, two are present in native apoflavodoxin as well. A substantial part of the third helix becomes beta-strand while forming native protein. Chemical shift changes due to amino acid residue replacement show that the latter alpha-helix has hydrophobic interactions with all other ordered regions in unfolded apoflavodoxin. Remarkably, these ordered segments dock non-natively, which causes strong competition with on-pathway folding. Thus, rather than directing productive folding, conformational preorganization in the unfolded state of an alpha-beta parallel-type protein promotes off-pathway species formation.

Accurate Random Coil Chemical Shifts from an Analysis of Loop Regions in Native States of Proteins

“…Figure S3), GED of dynamin in 9.7 M urea, 14 GED of dynamin in 6 M GuHCl, 15 SUMO from Drosophila melanogaster in 8 M urea, 16 and Azotobacter Vinelandii apoflavodoxin in 6 M GuHCl. 17 (Figure 2). To better account for sequence-dependent effects on chemical shifts, in principle, we could use amino acid triplets (or quintuples and so on); however, larger databases would be required to derive the corresponding parameters in these cases.…”

The ribosome regulates flavodoxin folding

“…α-Crystallin distinguishes between MG states of α-lactalbumin, and preferentially interacts with MGs on route to aggregation and precipitation (66). GroEL, sometimes in combination with GroES, recognizes MGs and may stimulate their productive folding, though the exact mechanisms by which it does this are still not completely understood (67)(68)(69). In addition, the ribosome seems to have a protein folding activity through Domain V rRNA (70).…”

“…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.…”

“…Intermediate Ion is an obligatory, high-energy on-pathway species, which is non-molten globular and rapidly converts to native apoflavodoxin (67). 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). 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.…”