Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin.
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.
Department of Agrotechnology and Food Sciences, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, NL-6703 HA Wageningen, The Netherlands.
Yves J M
YJ
Bollen
Ignacio E
IE
Sánchez
Carlo P M
CP
van Mierlo
D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D005418:Flavodoxin; C026932:apoflavodoxin; D019791:Guanidine