Formation of On- and Off-Pathway Intermediates in the Folding Kinetics of Azotobacter vinelandii Apoflavodoxin

Abstract

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.

The folding energy landscape of apoflavodoxin is rugged: Hydrogen exchange reveals nonproductive misfolded intermediates

“…where m N-U is the m value associated with global unfolding of apoflavodoxin (13,15) and m PUF-U is the one associated with unfolding of a specific PUF to unfolded apoflavodoxin. When ␣ ϭ 0, the corresponding PUF is as denaturant-accessible as fully unfolded apoflavodoxin, and when ␣ ϭ 1 it is as denaturantaccessible as native apoflavodoxin.…”

“…However, little evidence exists that links PUFs to the folding intermediates that populate during equilibrium unfolding, kinetic unfolding, or kinetic refolding of proteins. This missing link makes it difficult to position PUFs within the energy landscape for protein folding (10).Recently, both the denaturant-induced equilibrium unfolding and the kinetic folding of Azotobacter vinelandii flavodoxin have been characterized (11)(12)(13)(14)(15). Apoflavodoxin kinetic folding is described by (13) …”

“…Recently, both the denaturant-induced equilibrium unfolding and the kinetic folding of Azotobacter vinelandii flavodoxin have been characterized (11)(12)(13)(14)(15). Apoflavodoxin kinetic folding is described by (13) where U is unfolded protein, N is native apoflavodoxin, and I 1 and I 2 are folding intermediates.…”

“…Apoflavodoxin kinetic folding is described by (13) where U is unfolded protein, N is native apoflavodoxin, and I 1 and I 2 are folding intermediates. In the presence of FMN, first apoflavodoxin folds to its native state according to Eq.…”

“…The rate constants that describe departure from I 2 , either to N or to U, are not absolute. The kinetic data obtained only allow the determination of their ratio (13). The molten globule-like intermediate I 1 , which also populates to significant extents at GuHCl concentrations ranging from 1 to 3 M, acts as a trap.…”