Engineering the B1 domain of streptococcal protein G: Structural investigations by multidimensional heteronuclear NMR

The B1 domain of streptococcal protein G provides a well-characterized system for structural investigations of proteins. In this thesis, the urea-unfolded state has been characterized, the tolerance towards hydrophobic substitutions in the core has been surveyed, the hydrogen exchange behavior of the backbone amides has been elucidated, and structural information on a tetrameric mutant of this domain has been gathered.;The chemical shifts of the urea-unfolded state were assigned. The secondary chemical shifts, the 3JHNα coupling constants and the short-range NOEs gave no indication of residual structure. Measurement of the backbone 15N relaxation parameters revealed a region of restricted motion in the β3–β4 turn of the native protein. Motion in the rest of the protein was uniform, with the exception of 3–4 residues at either end of the chain.;A series of hydrophobic substitutions were made in the hydrophobic core. The resulting mutants were assayed for stability and overall fold. The core of the protein is particularly sensitive to substitutions at position 26. One of the mutants was unable to adopt the GB1 fold and optimized its stability by adopting a homo-tetrameric form.;Hydrogen exchange in the backbone amides was measured at 25°C. Rates of hydrogen exchange were inversely correlated with burial of the amide nitrogen. The slow-exchanging backbone amides did not correlate with the hydrogen bonds formed early in protein folding. Hydrogen exchange rates from NH to ND and from ND to NH were similar. The ratio between these two rates does not correlate with any obvious physical parameters of the hydrogen bonds.;Chemical shifts for the tetrameric mutant (HS...