Showing papers by "Jack H. Freed published in 2013"

Conformational ensemble of the sodium-coupled aspartate transporter

Abstract: The sodium and aspartate symporter GltPh mediates transport by alternating between outward-facing and inward-facing states. These conformational changes are now probed using double electron-electron resonance (DEER) spectroscopy. The data show that GltPh samples both states with similar probabilities, and that each protomer in the GltPh homotrimer behaves independently of the others.

Improved Sensitivity for Long-Distance Measurements in Biomolecules: Five-Pulse Double Electron−Electron Resonance

Abstract: We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron–electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER. The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 μM, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 μs, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both.

Pulse Dipolar Electron Spin Resonance: Distance Measurements

Abstract: In recent years electron spin resonance (ESR) has provided the means to obtain structural constraints in the field of structural biology on the nanoscale by measuring distances between paramagnetic species, which usually have been nitroxide spin-labels. These ESR methods enable the measurement of distances over the wide range from ca. 6–10 A to nearly 90 A. While cw methods may be used for the shortest distances, it is the pulse methods that enable this wide range, as well as determination of the distributions in distance. In this chapter we first describe the underlying theoretical concepts for understanding the principal pulse methods of double quantum coherence (DQC)-ESR and double-electron–electron-resonance (DEER), which we collectively refer to as Pulse-Dipolar ESR Spectroscopies (PDS). We then provide technical aspects of pulse ESR spectrometers required for high quality PDS studies. This is followed by an extensive description of sensitivity considerations in PDS, based largely upon our highly sensitive 17.3 GHz pulse spectrometer at ACERT. This description also includes a comparison of the effectiveness of the respective PDS pulse methods. In addition, the newer methods of 5-pulse DEER, which enables longer distances to be measured than by standard DEER, and 2D-DQC, which provides a convenient mapping for studying orientational coherence between spin labels and their interspin vector, are described.

HAMP domain conformers that propagate opposite signals in bacterial chemoreceptors

Abstract: HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between “on” and “off” conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.

DSC Characterization of Nematic-Smectic and Smectic-Smectic Phase Transitions in N(p-n-alkoxy benzilidene)-p-n-alkylanilines (n O. m)

Abstract: The DSC method of Navard and Haudin is applied to characterize the liquid-crystalline phase transitions in a series of n O.m compounds, which exhibit rich but subtle polymorphism. This method involves the measurement of the ratio N = h1/h (where h1 and h are the heights of DSC transition peaks at two heating rates, one being twice the other). The particular relevance to distinguishing the order of the nematic-smectic A (NA) phase transition is emphasized.