Random protein truncation

When structural information for a protein is lacking, identifying appropriate residues for mutational analysis is difficult. In such cases, random mutagenesis coupled with selection can provide a powerful means of analysis. We have used such an approach to examine how the seventeen C-terminal residues of BsCM contribute to enzyme efficiency [70], 

To address these questions, we developed a strategy involving random C-terminal truncation of the enzyme followed by selection of functional clones in the KA12/ 

Kinetic characterization of several selected BsCM variants shows that truncation or mutation of the C-terminal tail has little effect on the turnover number (fcc ll) of the enzyme (Tab. 3.1). When chorismate is bound to the active site of the variants, it is converted to prephenate nearly as efficiently as with wild-type BsCM. However, a substantial reduction in the k /K value is evident (Tab. 3.1). This finding indicates that the C-terminus, while not directly involved in the chemical transformation of bound ligand, does contribute to enzymatic efficiency by uniform binding of substrate and transition state. 

BsCM variant C-terminal amino acid sequence kal (S 1) W (M-1 s 1) 

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Figure 17.10 Construction of a two helix truncated Z domain, (a) Diagram of the three-helix bundle Z domain of protein A (blue) bound to the Fc fragment of IgG (green). The third helix stabilizes the two Fc-binding helices, (b) Three phage-display libraries of the truncated Z-domaln peptide were selected for binding to the Fc. First, four residues at the former helix 3 interface ("exoface") were sorted the consensus sequence from this library was used as the template for an "intrafece" library, in which residues between helices 1 and 2 were randomized. The most active sequence from this library was used as a template for five libraries in which residues on the Fc-binding face ("interface") were randomized. Colored residues were randomized blue residues were conserved as the wild-type amino acid while yellow residues reached a nonwild-type consensus, [(b) Adapted from A.C. Braisted and J.A. Wells,...

The first success was demonstration in 1994, with the report of a large, diverse library of decapeptides displayed and selected while associated with E. coli S30 polysomes and RNA.262 The key to Dower s success was the application of natural product antibiotics that were known to interfere with protein synthesis by stabilizing the ribosome-mRNA-protein complex. Thus, rifampicin and chloramphenicol (for prokaryotic system) or cycloheximide (for eukaryotic system) were used.2 3 Because these antibiotics halt the translation at random locations, the ensuing libraries were composed of mostly truncated peptides and thus not really suitable for the generation of cDNA libraries. Later, removal of the stop codon from mRNA was used to stall the translation at the end of the mRNA.264,265 Several improvements have been made more recently to stabilize the... 

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