Fluctuations of protein structure as expressed in the distribution of hydrogen exchange rate constants

Abstract

SynopsisHydrogen exchange kinetics of proteins provide information about the dynamics of their structure. T h e interpretation of experiments has been limited by dilf'iculty in identifying individual rate constants. In order to invert the kinetic data, and to gain inlormation about. the individual reacting sites, we introduce a Laplace inversion technique and obtain the distribution function of rate constants hy which the intricate reaction proceeds. A series of carefully overlapped experiments were performed on lysozyme a t %"C, an exchange profile obtained, and a distribution function extracted. This function was composed of a product of two terms, indicating two parallel pathways. T h e first, a power-law term, was attrihuted t o exchange from the native state. This part o f t h e distribution function thus describes the scope of the conformational fluctuations in proteins a t constant temperature and pressure. T h e second, a n exponential, was seen to be associated with the pathway involving thermal unfolding and subsequent free exchange with the solvent. The intluence of trichloroethanol and glycerol on the total distribution function was measured. Trichloroethanol selectively increased the contribution from the thermal unfolding pathway, whereas glycerol, hesides decreasing this type of Contribution, increased the width of the distribution function attributed to structural fluctuations.

The Complex Inter-Relationships Between Protein Flexibility and Stability

“…151 Similar conclusions were drawn based on isotope exchange experiments using azurin, myoglobin and lysozyme in the presence of glycerol. 149,150,[152][153][154] Carpenter and co-workers used FTIR spectroscopy to monitor the native state hydrogen exchange of three different proteins (RNase A, RNase S, and Cyt-C) in the presence and absence of sucrose. 147 In this study, sucrose was shown to increase the thermodynamic stability of all three proteins in a concentration dependent manner.…”

Study of Amide-proton Exchange of Escherichia coliMelibiose Permease by Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy

“…The analysis of hydrogen-deuterium exchange kinetics provides a means to gain further insight into the structure and dynamics of the protein, and to identify sub-molecular motional domains (22)(23)(24). The H/D exchange kinetics can be obtained by ATR-FTIR spectroscopy, applied to film samples.…”

Tritium exchange on transfer RNA: slowly exchanging protons sensitive to a change in the dihydrouridine stem.

“…Caution must nevertheless be exercised in interpreting the results of such a procedure (16,17). In fact, it is unlikely that the distribution of lifetimes is so discontinuous, especially in experiments on unfractionated tRNA.…”