Taking the Monte-Carlo gamble: How not to buckle under the pressure!

Consistent buckling distortions of a large membrane patch (200 × 200 Å) are observed during molecular dynamics (MD) simulations using the Monte-Carlo (MC) barostat in combination with a hard Lennard-Jones (LJ) cutoff. The buckling behavior is independent of both the simulation engine and the force field but requires the MC barostat-hard LJ cutoff combination. Similar simulations of a smaller patch (90 × 90 Å) do not show buckling, but do show a small, systematic reduction in the surface area accompanied by ~1 Å thickening suggestive of compression.

K channel C-type gating involves asymmetric selectivity filter order-disorder transitions.

K potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric Ks is unknown.

A kinetic mechanism for enhanced selectivity of membrane transport.

Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate.

A systems-biology approach to molecular machines: Exploration of alternative transporter mechanisms.

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal.

EmrE reminds us to expect the unexpected in membrane transport.

Grabe et al. celebrate a new mathematical model of the multidrug transporter EmrE, constructed from NMR and stop flow kinetic data.

Stochastic steps in secondary active sugar transport.

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function.

Structural determinants of water permeation through the sodium-galactose transporter vSGLT.

Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein.

The mechanism of sodium and substrate release from the binding pocket of vSGLT.

Membrane co-transport proteins that use a five-helix inverted repeat motif have recently emerged as one of the largest structural classes of secondary active transporters. However, despite many structural advances there is no clear evidence of how ion and substrate transport are coupled. Here we report a comprehensive study of the sodium/galactose transporter from Vibrio parahaemolyticus (vSGLT), consisting of molecular dynamics simulations, biochemical characterization and a new crystal structure of the inward-open conformation at a resolution of 2.

Water permeation through the sodium-dependent galactose cotransporter vSGLT.

It is well accepted that cotransporters facilitate water movement by two independent mechanisms: osmotic flow through a water channel in the protein and flow driven by ion/substrate cotransport. However, the molecular mechanism of transport-linked water flow is controversial. Some researchers believe that it occurs via cotransport, in which water is pumped along with the transported cargo, while others believe that flow is osmotic in response to an increase in intracellular osmolarity.

Automatic implementation of precise grid screens: the four-corners method.

Crystallization trials can be designed as a systematic gradient of the concentration of key reagents and/or pH centered on the original conditions. While the concept of the grid screen is simple, its implementation is tedious and difficult by hand. A procedure has been developed for preparing crystallization grid screens that is both efficient and achieves high accuracy because it relies on a limited number of solutions that are carefully prepared by hand.

Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease.

Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters.

Machine-learning techniques for macromolecular crystallization data.

Systematizing belief systems regarding macromolecular crystallization has two major advantages: automation and clarification. In this paper, methodologies are presented for systematizing and representing knowledge about the chemical and physical properties of additives used in crystallization experiments. A novel autonomous discovery program is introduced as a method to prune rule-based models produced from crystallization data augmented with such knowledge.

Adaptive integration method for Monte Carlo simulations.

We present an adaptive sampling method for computing free energies, radial distribution functions, and potentials of mean force. The method is characterized by simplicity and accuracy, with the added advantage that the data are obtained in terms of quasicontinuous functions. The method is illustrated and tested with simulations on a high density fluid, including a stringent consistency test involving an unusual thermodynamic cycle that highlights its advantages.

A kinesin switch I arginine to lysine mutation rescues microtubule function.

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated.

Motor domain mutation traps kinesin as a microtubule rigor complex.

Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu164 was identified as an amino acid critical for kinesin function [Brendza, K. M.