CORE MUTANTS OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN-G - STABILITY AND STRUCTURAL INTEGRITY

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 H-1-N-15 heteronuclear correlation NMR spectroscopy and fl uorescence. All mutants comprised changes in beta-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) formi ng the beta-sheet side of the sheet-helix core interface. A solvent ex posed position Ile-6 was chosen as a control. Randomization of bases a t codon positions 1 and 3 with thymine at position 2 introduces five p ossible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. T he distribution of encoded amino acids at all five positions is approx imately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integr ity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutan ts is not strictly correlated with the number of changes or residue vo lume.