A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H-15N heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in beta-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the beta-sheet side of the sheet-helix core interface. A solvent exposed position Ile-6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume. Study holds ProTherm entries: 9794, 9795, 9796, 9797, 9798, 9799, 9800 Extra Details: protein G; B1 domain; core mutant; library; protein design;,structure; stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||21|
|Proteins||Immunoglobulin G-binding protein G ; Immunoglobulin G-binding protein G|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE|