Protein hydrogen exchange mechanism: local fluctuations

Abstract

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This "local fluctuation" mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix.

Figure 1.

Unfolding free energy from GdmCl melting (oxidized Cyt c, 0.5 M KCl, 20°C). The Lys8Gly mutation is destabilizing by 1.4 kcal/mole across the pH range used in the HX experiments. (Stability increases by 0.45 kcal/mole in D₂O where HX NMR measurements were made.)

Figure 2.

ΔG°_ex values calculated for the determining opening reactions from HX rates (equations 1, 3, 4; 0.5 M KCl, 20°C), using k_int calibrated for unstructured molecules, usually averaged from experiments at more than one pH. (Use of the ΔG°_ex parameter is convenient but may not accurately represent the free energy of the structural distortion; see Discussion).

Figure 3.

HX rate changes. (A) The HX rate ratio, k_K8G/k_pWT, for residues that could be measured in both pWT and Lys8Gly proteins. The ratio is relatively independent of k_int except for residue 8 itself, where a factor of 2 enters (not applied in the graph shown). (B) HX data for residues near Lys8 (closed symbols) or Gly 8 (open symbols), measured at pH* 6 or corrected to pH* 6 when measured at another pH* (residue 8 at pH* 5.3; residue 10 at pH* 8.4).

Figure 3.

HX rate changes. (A) The HX rate ratio, k_K8G/k_pWT, for residues that could be measured in both pWT and Lys8Gly proteins. The ratio is relatively independent of k_int except for residue 8 itself, where a factor of 2 enters (not applied in the graph shown). (B) HX data for residues near Lys8 (closed symbols) or Gly 8 (open symbols), measured at pH* 6 or corrected to pH* 6 when measured at another pH* (residue 8 at pH* 5.3; residue 10 at pH* 8.4).