Biophysical Interactions of the OHC Motor Protein Prestin: A Study at the Single Molecule Level

The exquisite frequency selectivity and amplification characteristics of mammalian hearing intimately depend on the fast electromechanical motion of the outer hair cells in the cochlea. This membrane based process, termed electromotility, is driven by the protein prestin which is uniquely present in the OHC lateral wall. Voltage dependent motility, in OHCs and mammalian cells expressing prestin, is accompanied by intramembranous charge movement which is widely considered a signature of electromotility and prestin function. How prestin converts changes in membrane potential into axial length changes of OHCs is currently not understood at the molecular level. Many electromotility models predict that prestin conformational changes are the underlying mechanism connecting charge movement and motility. Currently, however, only indirect evidence for a prestin conformational change is available. Various experiments have indicated that the oligomeric states of prestin may be an important determinant of function. Numerous reports have provided varying estimates of prestin oligomeric state. However, estimates have been based on measurements performed outside the membrane making, firm biophysical conclusions difficult. Biophysical studies of prestin function have demonstrated its dependence on membrane properties. Alterations of membrane cholesterol affect voltage dependence of charge movement and motility. In addition cholesterol manipulations cause spatial redistribution of prestin and possibly change prestin oligomeric state. However, the underlying cause for prestin sensitivity to cholesterol and its relation to membrane distribution is unknown. We have applied single molecule fluorescence (SMF) imaging, single particle tracking (SPT), and Förster resonance energy transfer (FRET) to investigate prestin interactions at the molecular level. The results of our SMF experiments have suggested that prestin forms mainly tetramers and dimers in the cell membrane. Using SPT to map the trajectories of prestin in the membrane, we have found that prestin undergoes diffusion in and hops between membrane confinements of varying size. In addition, we have found that cholesterol affects the size and confinement strength of the compartments but does not affect the diffusivity within the compartments. Finally, using a combination of electrophysiology and FRET we have demonstrated that prestin undergoes voltage dependent structural changes. In total, our results refine our molecular understanding of prestin function

Response to Comment on "Pairing and Phase Separation in a Polarized Fermi Gas"

Zwierlein and Ketterle rely on subjective arguments and fail to recognize important differences in physical parameters between our experiment and theirs. We stand by the conclusions of our original report

Pairing and Phase Separation in a Polarized Fermi Gas

We report the observation of a pairing in a number polarized two-component gas of atomic fermions. Beyond a critical polarization, the gas separates into a superfluid paired core surrounded by a shell of normal unpaired fermions. The critical polarization diminishes with decreasing attractive interaction. We also measure the parameter \beta = 0.54 (5) describing the universal energy of a strongly interacting Fermi gas, and find good agreement with most recent theory. These results are relevant to predictions of exotic new phases of quark matter and of strongly magnetized superconductors

Measurements of the interactions in a paired zero temperature lithium-6 gas throughout the BEC-BCS crossover

Understanding the effects of interactions on the ground-state of a paired degenerate Fermi gas throughout the BEC-BCS crossover has been a long standing physical quest for which countless papers have been written. The crossover describes the smooth transition of the physics of a BEC of tightly bound dimers to that of a paired BCS superfluid. A Feshbach resonance is used to tune the interactions necessary to study the crossover. Right around resonance the interactions are expected to be parameterized by a single universal parameter beta [1--3]. This thesis describes a measurement of the axial size of paired, 6Li clouds in the BEC-BCS crossover and provides a comparison with theory. In the BEC regime, absolute measurements of the molecular scattering length are compared with the atomic scattering length and the ratio is compared with theory. Finally, a measurement of beta is made and compared with theory

Facilitated dissociation of transcription factors from single DNA binding sites

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate [Formula: see text], establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap

Facilitated dissociation of transcription factors from single DNA binding sites

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate [Formula: see text], establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap