Hydrogen-Deuterium Exchange in the Free and Immunoglobulin G-Bound Protein G B-Domain

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Abstract

Hydrogen-deuterium exchange experiements have been used to measure backbone amide proton (NH) exchange rates in the free and IgG-bound protein G B2-domain (GB2). Exchange rates were analyzed in terms of the free energy required for transient opening of an H-bonded NH (delta Gop), and exchange mechanisms were interpreted in the context of local and global opening motions. In free GB2 at 22 degrees C, 28 detectable NHs have delta Gop values which approximate the free energy of thermal unfolding (delta Gu) 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 Gop values, consistent with a less cooperative exchange mechanism than in free GB2. The large range of protection factors (1.3 to > 210) also indicates that exchange does not occur cooperatively for all detectable NHs in bound GB2. 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 approximately 10(8) M-1. The pattern of high protection observed in the helix overlaps with the putative binding site suggested from previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

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