HYDROGEN-DEUTERIUM EXCHANGE IN THE FREE AND IMMUNOGLOBULIN G-BOUND PROTEIN-G B-DOMAIN

Hydrogen-deuterium exchange experiments have been used to measure backbone amide proton (NH) exchange rates in the free and IgG-bound protein G B2-domain (G(B2)). Exchange rates were analyzed in terms of the free energy required for transient opening of an H-bonded NH (Delta G(op)), and exchange mechanisms were interpreted in the context of local and global opening motions. In free G(B2) at 22 degrees C, 28 detectable NHs have Delta G(op) values which approximate the free energy of thermal unfolding (Delta G(B)) obtained from calorimetry. This indicates that the majority of detectable NHs exchange through a global unfolding mechanism, reflecting the cooperative two-state unfolding behavior observed thermodynamically [Alexander et al. (1992) Biochemistry 31, 3597-3603]. IgG binding results in a broadening of exchange rates and Delta G(op) values, consistent with a less cooperative exchange mechanism than in free G(B2). The large range of protection factors (1.3 to >210) also indicates that exchange does not occur cooperatively for all detectable NHs in bound G(B2). Nineteen of the detectable NHs have significantly slowed exchange rates in the complex with protection factors >5. Residues with protection factors of the order of 100 or more occur in both the helix region (F30, K31, A34) and in the central core of the beta-sheet (V6, F52, V54). The highest protection factors are consistent with a binding constant of similar to 10(8) M(-1). The pattern of high protection observed in the helix overlaps with the putative binding site suggested from previous studies. However, the highly protected residues in the central core of the beta-sheet are removed from the putative binding interface. This suggests that IgG binding affects conformational dynamics in G(B2). The highly protected residues, which are mostly buried from the solvent-accessible surface, constitute the slow-exchange core of bound G(B2) and most likely exchange as a cooperative unit by global unfolding. Thus, the rigid slow-exchange core probably corresponds with the folding core of the bound protein. Residues with intermediate protection factors are situated further out from the slow-exchange core, and the smallest protection factor residues are near the surface or at the ends of beta-strands. These latter residues can exchange in the complex without complete unfolding of bound G(B2). While these locally unfolded states are still energetically costly, they do not greatly disrupt IgG binding. However, globally unfolded states are not consistent with binding to IgG.