Thermodynamics of structural fluctuations in lysozyme as revealed by hydrogen exchange kinetics

Abstract

A new method is described that makes use of the empirical enthalpy--entropy compensation behavior of a related series of processes for deriving the activation enthalpy and entropy probability density functions from the corresponding rate constant density function. The method has been applied to data obtained from a study of the temperature dependence of hydrogen-tritium exchange in lysozyme. Analysis of the temperature dependence of tj, the time required to reach a particular number of hydrogens remaining unexchanged, provides estimates of delta G#, delta H#, and delta S# for the exchange process. The results are consistent with the notion of two mechanisms of exchange characterized by different activation energies. Increases in delta H# are compensated by corresponding increases in delta S#. The compensation plot, however, reveals two distinct apparent compensation temperatures, which reflect the operation of two qualitatively different mechanisms of exchange. The faster hydrogens exchange with delta H# values between 8 and 18 kcal X mol-1 and are characterized by a high compensation temperature of 470 K. The slower hydrogens exchange with delta H# values that reach 40 kcal X mol-1 and display a compensation temperature of congruent to 360 K. The latter is associated with a thermal unfolding mechanism of exchange.

Hydrogen exchange and structural dynamics of proteins and nucleic acids

“…(9.1.5) Exchange at high pH BPTI presents further challenges at higher pH. In the pH range around 8, significant flattening in the rising rate curve (at 51 °C) occurs for residues 22 and 23 Woodward & Hilton, 1980) and perhaps also 21 (see analysis by Gregory et al 1982). This has been interpreted by Hilton and Woodward in terms of an EXi plateau.…”

“…When pH is adjusted to minimize peptide NH exchange rate (c. pH 3, see Fig. 8a), apparent water catalysis may appear (Gregory et al 1982). The above principles suggest that this may be due not to direct transfer to water molecules acting as a general base acceptor but rather to a more interesting mechanism involving pre-equilibrium protonation at the peptide carbonyl (/>K a about -1) and subsequent deprotonation by hydroxide.…”

“…(An analogous pH-independent mechanism dominates the H-exchange behaviour of the exocyclic amino protons in the nucleic acids (Section 7.3).) According to Gregory et al (1982) the apparent peptide group water catalysis has a relatively high temperature dependence (21 kcal/mol), so that the pH-independent contribution to protein H-exchange rate should become more apparent at elevated temperatures, especially in the region of pH mln , where specific acid and base catalysis are at a minimum. However, the significance of this effect is in some doubt since a study of H-exchange rates in a destabilized, completely unfolded BPTI derivative at 8o° (Roder et al 1983 b) shows simple pH dependence according to equation (5.5).…”

“…(3) exchange is EX2, but K op and k ch have equal and opposite dependence on pH. Gregory et al (1982) have reported apparent water catalysis of peptide NH exchange in some random-chain polypeptides. This reaction appears to have relatively high activation energy, so that its contribution to HX pathways should become more prominent with increasing temperature.…”

Local Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody

“…Proteins such as immunoglobulins can exhibit motions with different magnitudes of spatial and temporal scales, which may arise from distinct regions within the protein. The thermodynamic and kinetic implications of such distinct dynamic regions in proteins have been previously studied 16–18. These regions may have different degrees of exposure to the solvent in their immediate vicinity primarily involving water of hydration.…”