Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin.
Flavodoxin is an alpha/beta protein with a noncovalently bound flavin-mononucleotide (FMN) cofactor. The apo-protein adopts a structure identical to that of the holo-form, although there is more dynamics in the FMN-binding loops. The equilibrium unfolding processes of Azotobacter vinelandii apo-flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo- and holo-flavodoxins involve rather stable intermediates. In contrast, we here show that both holo- and apo-forms of flavodoxin from D. desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two-state equilibrium processes. Moreover, the FMN cofactor remains bound to the unfolded holo-protein. The folding and unfolding kinetics for holo-flavodoxin exhibit two-state behavior, albeit an additional slower phase is present at very low denaturant concentrations. The extrapolated folding time in water for holo-flavodoxin, approximately 280 microsec, is in excellent agreement with that predicted from the protein's native-state topology. Unlike the holo-protein behavior, the folding and unfolding reactions for apo-flavodoxin are best described by two kinetic phases, with rates differing approximately 15-fold, suggesting the presence of a kinetic intermediate. Both folding phases for apo-flavodoxin are orders of magnitude slower (40- and 530-fold, respectively) than that for the holo-protein. We conclude that polypeptide-cofactor interactions in the unfolded state of D. desulfuricans strain 29577 flavodoxin alter the kinetic-folding path towards two-state and speed up the folding reaction.
Apiyo
David
D
Chemistry Department, Tulane University, 6823 St Charles Avenue, New Orleans, LA 70118, USA.
Wittung-Stafshede
Pernilla
P
D003901:Desulfovibrio / Q000378:metabolism*; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D005418:Flavodoxin; D005486:Flavin Mononucleotide; D019791:Guanidine