Solvation Information

Upon exposure to D2O, the amide proton of the protein backbone will exchange with the solvent deuterium. The H/D exchange rate depends upon pH, temperature and the structural environment of the amide proton (Gregory and Lumry 1985). Factors such as steric inaccessibility to the solvent, local charge distributions and internal hydrogen bonding will slow down the rate of amide proton exchange so that the kinetics of D2O diffusion inside the protein core probes the accessibility of the solvent into random or structured internal domains and thus can give information on the protein tertiary structure. NMR or mass spectrometry and isotope-exchange techniques can be used to probe the tertiary structure or conformational stability of proteins (Huyghues-Despointes et al. 2001). For instance, these techniques were carried out for lysozyme before and after elution from reversed chromatographic phases (McNay and Fernandez 1999; Tibbs Jones and Fernandez 2003). The mass increase in the protein resulting from H/D exchange may be measured by mass spectrometry. For example, localised changes in the structural stability of myoglobin upon adsorption onto nanometer-sized silica particles have been studied with isotopic exchange mass spectrometry (Buijs et al. 2003).

Exchange-rate measurements can be performed directly on adsorbed proteins by infrared spectroscopy from the decrease of the so-called amide II absorption band as a function of pH (Servagent-Noinville et al. 2000). The amide II absorption band corresponds to the bending motions of the residual CONH groups resulting from the incubation of the protein in deuter-ated buffer. Two-dimensional (2D)-FTIR spectroscopy developed by Noda (1989) could be used to enhance the spectral resolution of both the amide I and II bands of proteins and to allow assignments of the conformation-sensitive component bands (Nabet and Pezolet 1997). Two-dimensional FTIR spectroscopy combined with isotopic exchange has revealed the assignment of the exchanging amide proton to changes in protein secondary structure induced by a ligand binding (Meskers et al. 1999). Surface-induced conformational changes of cytochrome c and of antimicrobial peptides have also been demonstrated by the use of 2D-FTIR spectroscopy (Lecomte et al. 2001; Noinville et al. 2003).

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