Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif.


Abstract

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alphabeta motif (CS alphabeta) consists of an alpha-helix and an antiparallel triple-stranded beta-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CS alphabeta motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CS alphabeta motif. The information is valuable to the scaffold with the CS alphabeta motif and protein engineering. Study holds ProTherm entries: 25234, 25235, 25236, 25237, 25238, 25239, 25240, 25241, 25242, 25243, 25244, 25245, 25246, 25247, 25248, 25249, 25250, 25251, 25252, 25253, 25254, 25255, 25256, 25257, 25258, 25259, 25260, 25261, 25262, 25263, 25264, 25265, 25266 Extra Details: 1mM EDTA was added in the experiment plant defensin; alpha-amylase; alanine scan; circular dichroism spectroscopy; fluorescence spectroscopy; protein engineering

Submission Details

ID: xyn9MtLQ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Yang YF;Cheng KC;Tsai PH;Liu CC;Lee TR;Lyu PC,Protein Sci. (2009) Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif. PMID:19533758
Additional Information

Study Summary

Number of data points 33
Proteins plant defensin ; Plant defensin
Unique complexes 33
Assays/Quantities/Protocols Experimental Assay: Cm
Libraries Mutations for sequence RTCMIKKEGWGKCLIDTTCAHSCKNRGYIGGNCKGMTRTCYCLVNC

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)