Conformation And Dynamics Of A Rhodamine Probe Attachedat Two Sites On A Protein: Implications For Molecularrnstructure Determination In Situ

摘要

Replica exchange molecular dynamics (REMD) calculations were used to determine the conformation and dynamics of bifunctional rhodamine probes attached to pairs of cysteines in three model systems: (a) a polyalanine helix, (b) the isolated C helix (residues 53-66) of troponin C, and (c) the C helix of the N-terminal region (residues 1-90) of troponin C (sNTnC). In each case, and for both diastereoisomers of each probe-protein complex, the hydrophobic face of the probe is close to the protein surface, and its carboxylate group is highly solvated. The visible-range fluorescence dipole of the probe is approximately parallel to the line joining the two cysteine residues, as assumed in previous in situ fluorescence polarization studies. The independent rotational motion of the probe with respect to the protein on the nanosecond time scale is highly restricted, in agreement with data from fluorescence polarization and NMR relaxation studies. The detailed interaction of the probe with the protein surface depends on steric factors, electrostatic and hydrophobic interactions, hydrogen bonds, and hydration effects. The interaction is markedly different between diastereoisomers, and multiple preferred conformations exist for a single diasteroisomer. These results show that the combination of the hydrophobic xanthylium moiety of bifunctional rhodamine with the carboxylate substitution in its pendant phenyl ring causes the probe to be immobilized on the protein surface, while the two-site cysteine attachment defines the orientation of its fluorescence dipole. These features allow the orientation of protein components to be accurately determined in situ by polarized fluorescence measurements from bifunctional rhodamine probes.