Specific interaction of the Streptomyces chitin-binding protein CHB1 with alpha-chitin--the role of individual tryptophan residues.
Streptomyces olivaceoviridis secretes a so far unique protein of 18.7 kDa (CHB1) which lacks catalytic activity. It interacts highly specifically with alpha-chitin, but not with beta-chitin, chitosan, or cellulose. Each of the five codons for tryptophan (Trp) in the chb1 gene was replaced by those for leucine (Leu) or tyrosine (Tyr). Eight corresponding mutant proteins and the wild-type protein were purified to homogeneity and their binding capacity to alpha-chitin was determined. The relative affinities to anti-CHB1 antibodies, the kinetics of binding, the dissociation constants, circular dichroism, and fluorescence emission spectra for three mutant types were compared to the characteristics of CHB1. The presented data lead to the following conclusions. (a) CHBI presents a highly flexible protein lacking alpha-helices. (b) Replacement of each of the buried Trp residues (Trp134 and Trp184) leads to conformational alterations and, in due course, to a considerably reduced binding affinity of the protein. (c) The exchange of the exposed Trp 57 by either Leu or Tyr results in relatively slight topological changes, but entails a loss of binding capacity of about 90%. (d) The dissociation constant was highest for the mutant protein [L57]CHB1 (2.17 microM), followed by [L134]CHB1 (0.91 microM) and [L184]CHB1 (0.26 microM), and lowest for the progenitor CHB1 (0.11 microM), indicating its strong affinity to the unsoluble substrate. (e) The data suggest that the exposed Trp57 contributes directly and significantly to the interaction of CHB1 with alpha-chitin.
Zeltins
A
A
FB Biologie/Chemie, University of Osnabrück, Germany.
Schrempf
H
H
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D013302:Streptomyces / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D001426:Bacterial Proteins; C091405:CHB1 protein, Streptomyces; D002352:Carrier Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D002686:Chitin; D014364:Tryptophan