Formation of on- and off-pathway intermediates in the folding kinetics of azotobacter vinelandii apoflavodoxin
| Autor: | Yves J. M. Bollen, Ignacio E. Sánchez, Carlo P. M. van Mierlo |
|---|---|
| Přispěvatelé: | Structural Biology |
| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Předmět: |
Protein Denaturation
Protein Folding binding Protein Conformation barriers Population Flavodoxin Biochemie mechanism Biochemistry Peptide bond Denaturation (biochemistry) SDG 7 - Affordable and Clean Energy education Protein secondary structure lysozyme Guanidine VLAG education.field_of_study Azotobacter vinelandii biology Chemistry transition secondary structure dynamics biology.organism_classification Molten globule Chevron plot Crystallography Kinetics Spectrometry Fluorescence protein structures Protein folding fluorescence Apoproteins intensity Signal Transduction |
| Zdroj: | Biochemistry 43 (2004) 32 Bollen, Y J M, Sanchez, I E & Van Mierlo, C P M 2004, ' Formation of on-and off-pathway intermediates in the folding kinetics of azotobacter vinelandii apoflavodoxin. ', Biochemistry, vol. 43, pp. 10475-10489 . https://doi.org/10.1021/bi049545m Biochemistry, 43(32), 10475-10489 Biochemistry, 43, 10475-10489. American Chemical Society |
| ISSN: | 0006-2960 |
| Popis: | 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 double left right arrow U double left right arrow I-on double left right arrow 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 |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|