Benjamin C Stevens | PubMed Health

PubMed2006

Bridging conformational dynamics and function using single-molecule spectroscopy.

In a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the "structure-function relation" by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase.

Adenosine Triphosphate↗

PubMed2004

Discrete and heterogeneous rotational dynamics of single membrane probe dyes in gel phase supported lipid bilayer.

In order to probe the local dynamics of lipid bilayers in the gel phase, we measured the rotational time trajectories of a membrane probe, diI(3), in supported bilayers of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) using single molecule fluorescence polarization imaging. diI(3) has two hydrocarbon tails that mimic phospholipid tails and has its transition dipole moment lying mostly on the plane of the membrane; hence it is an excellent probe for rotational dynamics in membranes. Above the transition temperature, the probes are laterally mobile and do not display polarized emission. In the gel phase below the transition temperature, lateral mobility is severely reduced and the emission becomes polarized with its polarization direction changing in the milliseconds time scale. Molecule by molecule analysis of the rotational time scales revealed significant heterogeneities among molecules, much larger than would be due to statistical noise. Control experiments using small unilamellar vesicles suggest that the heterogeneities are not caused by surface interactions and are intrinsic to the gel phase membrane. The rotational dynamics is strongly temperature dependent and the thermally activated state for the rotational motion has a large entropic barrier (> 30kB), indicating that relatively large local disorder is required for the rotational motion to occur. Rotational hopping between discrete angles has been observed at the lowest temperatures (approximately 10 degrees C). Our results suggest that the gel phase membrane is not uniform at the microscopic level but is highly dynamic with the rigidity of local environments constantly changing.

Carbocyanines↗