Long-Range Coupling in an Allosteric Receptor Revealed by Mutant Cycle Analysis

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50–60 Å from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (βL9′S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC50 measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors

Total angular momentum representation for atom-molecule collisions in electric fields

It is shown that the atom-molecule collision problem in the presence of an external electric field can be solved using the total angular momentum representation in the body-fixed coordinated frame, leading to a computationally efficient method for ab initio modeling of low-temperature scattering phenomena. Our calculations demonstrate rapid convergence of the cross sections for vibrational and Stark relaxation in He-CaD collisions with the number of total angular momentum states in the basis set, leading to a 5-100 fold increase in computational efficiency over the previously used methods based on the fully uncoupled space-fixed representation. These results open up the possibility of carrying out numerically converged quantum scattering calculations on a wide array of atom-molecule collisions and chemical reactions in the presence of electric fields.Comment: 19 pages, 3 figures, 1 tabl

Voltage-dependent Block of the Cystic Fibrosis Transmembrane Conductance Regulator Cl- Channel by Two Closely Related Arylaminobenzoates

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 µM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses ~40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 ± 0.4 pS in symmetric 150 mM Cl^-. A subconductance state, measuring ~60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 µM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at V_m =-100 mV and not at V_m = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (~ 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at V_m = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway

SUSY and Dark Matter Constraints from the LHC

The ability of the LHC to make statements about the dark matter problem is considered, with a specific focus on supersymmetry. After reviewing the current strategies for supersymmetry searches at the LHC (in both CMS and ATLAS), some key ATLAS studies are used to demonstrate how one could establish that SUSY exists before going on to measure the relic density of a neutralino WIMP candidate. Finally, the general prospects for success at the LHC are investigated by looking at different points in the MSSM parameter space.Comment: Talk given at the XLIrst Rencontres de Moriond session devoted to Electroweak Interactions And Unified Theories in March 2006, to be published in the associated proceedings. 10 pages, 8 figure

Fast Simulation of Facilitated Spin Models

We show how to apply the absorbing Markov chain Monte Carlo algorithm of Novotny to simulate kinetically constrained models of glasses. We consider in detail one-spin facilitated models, such as the East model and its generalizations to arbitrary dimensions. We investigate how to maximise the efficiency of the algorithms, and show that simulation times can be improved on standard continuous time Monte Carlo by several orders of magnitude. We illustrate the method with equilibrium and aging results. These include a study of relaxation times in the East model for dimensions d=1 to d=13, which provides further evidence that the hierarchical relaxation in this model is present in all dimensions. We discuss how the method can be applied to other kinetically constrained models.Comment: 8 pages, 4 figure

Dynamic facilitation explains democratic particle motion of metabasin transitions

Transitions between metabasins in supercooled liquids seem to occur through rapid "democratic" collective particle rearrangements. Here we show that this apparent homogeneous particle motion is a direct consequence of dynamic facilitation. We do so by studying metabasin transitions in facilitated spin models and constrained lattice gases. We find that metabasin transitions occur through a sequence of locally facilitated events taking place over a relatively short time frame. When observed on small enough spatial windows these events appear sudden and homogeneous. Our results indicate that metabasin transitions are essentially "non-democratic" in origin and yet another manifestation of dynamical heterogeneity in glass formers.Comment: 6 pages, 6 figure