2D-IR spectroscopy of the sulfhydryl band of cysteines in the hydrophobic core of proteins.
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 alpha-helix. The vibrational lifetime of the sulfhydryl group (T1 approximately 6 ps) is considerably longer than that of the much more commonly used amide I mode (approximately 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.
Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
M
M
Koziński
S
S
Garrett-Roe
P
P
Hamm
D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D006454:Hemoglobins; D013438:Sulfhydryl Compounds; D003545:Cysteine