Apoflavodoxin folding mechanism: an alpha/beta protein with an essentially off-pathway intermediate.
The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization. Unlike many cases where the presence of an intermediate has been detected either by a burst phase or by the curvature, at low urea concentration, of the otherwise only observable kinetic phase, two kinetic phases are observed in apoflavodoxin folding whose total amplitude equals the amplitude of unfolding. To determine the role of the intermediate in the folding reaction, the apoflavodoxin kinetic data have been fitted to all conceivable three-species kinetic models (either linear or triangular). Using a stepwise fitting procedure, we find that the off-pathway mechanism explains most of the kinetic data (not a slow unfolding phase), the on-pathway mechanism being rejected. By using global analysis, good overall agreement between data and fit is found when a triangular mechanism is considered. The fitted values of the microscopic constants indicate that most of the unfolded molecules refold from the denatured state. Apoflavodoxin thus folds via a triangular, but essentially off-pathway, mechanism. We calculate that the retardation of the folding caused by the off-pathway intermediate is not large. Some unusual properties of the intermediate are discussed.
Fernández-Recio
J
J
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009-Zaragoza, Spain.
Genzor
C G
CG
Sancho
J
J
D017033:Anabaena / Q000737:chemistry / Q000235:genetics; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004355:Drug Stability; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D001059:Apoproteins; D001426:Bacterial Proteins; D005418:Flavodoxin; D011994:Recombinant Proteins; C026932:apoflavodoxin; D011392:Proline