Hydrogen-deuterium (H-D) exchange experiments have been used to measure exchange rates for almost all of the main-chain amide protons (NHs) in the B1 and B2 IgG-binding domains of protein G. For H-bonded NHs, exchange rates were also measured as a function of temperature from 25 to 65 degrees C for B1 and from 25 to 60 degrees C for B2. A number of NHs exchange by a mechanism consistent with global unfolding. For these residues, the free energy required for transient opening of a H-bonded NH (delta Gop) from H-D exchange approximates the extrapolated free energy of thermal unfolding (delta Gu) from calorimetry in B1 and B2. The difference in exchange rates between B1 and B2 for these residues reflects the 1 kcal mol-1 difference in stability from calorimetry. The more stable B1 domain appears to have a slightly larger core of residues which exchange by global unfolding than B2. The delta Gop values for slow exchange H-bonded NHs and calorimetric delta Gu provide highly complementary information on the delta G versus temperature stability profiles of B1 and B2. Furthermore, NH exchange rates provide a very sensitive measure of local stability differences between B1 and B2. In both domains, the beta 2-strand is the least stable of the beta-sheet although it is more stable in B1 than B2. The largest local stability differences occur at residues Y3 and T18 which exchange 40-fold and 100-fold slower in B1, respectively. These residues form a H-bond donor-acceptor pair at one end of the beta 1-beta 2 region. Local stability differences are also evident near the beta 1-beta 2 turn. These stability differences are, at least in part, due to subtle differences in hydrophobic packing effects. They are not obvious from inspection of the B1 and B2 structures but are manifested as readily measurable changes in NH exchange rates for individual residues. Knowledge of these local stability differences in the beta 1-beta 2 region provides potential approaches for designing new stability mutants in protein G. Most non-H-bonded NHs have exchange rates that are < 15 times slower than their intrinsic rates. In marked contrast, the NHs of K10, T11, and L12 in the beta 1-beta 2 turn and V21 have exchange rates which are five to > or = 24 times faster than their intrinsic rates, probably due to local electrostatic effects.(ABSTRACT TRUNCATED AT 400 WORDS)