Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity.
Escherichia coli FtsH, which belongs to the ATPases associated with diverse cellular activities (AAA) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. To elucidate the molecular mechanisms of the proteolysis, a system for precisely monitoring substrate degradation is required. We have exploited E. coli flavodoxin containing non-covalently bound flavin mononucleotide (FMN) as a model substrate for monitoring protein degradation. It was found that FtsH degrades FMN-free apo-flavodoxin but not holo-flavodoxin. However, degradation of a mutant flavodoxin carrying a substitution of Tyr94 to Asp with a lower affinity for FMN could be monitored by fluorimetry. This newly developed monitoring system will also be applicable for proteolysis by other ATP-dependent proteases.
Okuno
Takashi
T
Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
Yamanaka
Kunitoshi
K
Ogura
Teru
T
D049069:ATP-Dependent Proteases / Q000378:metabolism*; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005470:Fluorometry; D005982:Glutathione Transferase / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D011993:Recombinant Fusion Proteins; D001216:Asparagine; D005486:Flavin Mononucleotide; D005982:Glutathione Transferase; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli