Abstract

We investigate the sulfhydryl band of cysteines as a new chromophore for two-dimensional IR (2D-IR) studies of the structure and dynamics of proteins. Cysteines can be put at almost any position in a protein by standard methods of site-directed mutagenesis and, hence, have the potential to be an extremely versatile local probe. Although being a very weak absorber in aqueous environment, the sulfhydryl group gets strongly polarized when situated in an alpha-helix inside the hydrophobic core of a protein because of a strong hydrogen bond to the backbone carbonyl group. The extinction coefficient (epsilon = 150 M-1 cm(-1)) then is sufficiently high to perform detailed 2D-IR studies even at low millimolar concentrations. Using porcine (carbonmonoxy)hemoglobin as an example, which contains two such cysteines in its wild-type form, we demonstrate that spectral diffusion deduced from the 2D-IR line shapes reports on the overall-breathing of the corresponding (x-helix. The vibrational lifetime of the sulfhydryl group (T-1 approximate to 6 ps) is considerably longer than that of the much more commonly used amide I mode (approximate to 1.0 ps), thereby significantly extending the time window in which spectral diffusion processes can be observed. The experiments are accompanied by molecular dynamics simulations revealing a good overall agreement.