Mutation analysis of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.
Cellulose-binding protein A (CbpA) has been previously shown to mediate the interaction between crystalline cellulose substrates and the cellulase enzyme complex of Clostridium cellulovorans. CbpA contains a family III cellulose-binding domain (CBD) which, when expressed independently, binds specifically to crystalline cellulose. A series of N- and C-terminal deletions and a series of small internal deletions of the CBD were created to determine whether the entire region previously described as a CBD is required for the cellulose-binding function. The N- and C-terminal deletions reduced binding affinity by 10- to 100-fold. Small internal deletions of the CBD resulted in substantial reduction of CBD function. Some, but not all, point mutations throughout the sequence had significant disruptive effects on the binding ability of the CBD. Thus, mutations in any region of the CBD had effects on the binding of the fragment to cellulose. The results indicate that the entire 163-amino-acid region of the CBD is required for maximal binding to crystalline cellulose.
Section of Molecular and Cellular Biology, University of California, Davis 95616.
M A
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Goldstein
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Doi
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
C085674:CbpA protein, E coli; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D002482:Cellulose