Finite Temperature String Method with Umbrella Sampling: Application on a Side Chain Flipping in Mhp1 Transporter

Protein conformational change is of central importance in molecular biology. Here we demonstrate a computational approach to characterize the transition between two metastable conformations in all-atom simulations. Our approach is based on the finite temperature string method, and the implementation is essentially a generalization of umbrella sampling simulations with Hamiltonian replica exchange. We represent the transition pathway by a curve in the conformational space, with the curve parameter taken as the reaction coordinate. Our approach can efficiently refine a transition pathway and compute a one-dimensional free energy as a function of the reaction coordinate. A diffusion model can then be used to calculate the forward and backward transition rates, the major kinetic quantities for the transition. We applied the approach on a local transition in the ligand-free Mhp1 transporter, between its outward-facing conformation and an intermediate conformation with the side chain of Phe305 flipped to the outside of the protein. Our simulations predict that the flipped-out position of this side chain has a free energy 6.5 kcal/mol higher than the original position in the crystal structure, and that the forward and backward transition rates are in the millisecond and submicrosecond time scales, respectively

Microspinning: Local Surface Mixing via Rotation of Magnetic Microparticles for Efficient Small-Volume Bioassays

The need for high-throughput screening has led to the miniaturization of the reaction volume of the chamber in bioassays. As the reactor gets smaller, surface tension dominates the gravitational or inertial force, and mixing efficiency decreases in small-scale reactions. Because passive mixing by simple diffusion in tens of microliter-scale volumes takes a long time, active mixing is needed. Here, we report an efficient micromixing method using magnetically rotating microparticles with patterned magnetization induced by magnetic nanoparticle chains. Because the microparticles have magnetization patterning due to fabrication with magnetic nanoparticle chains, the microparticles can rotate along the external rotating magnetic field, causing micromixing. We validated the reaction efficiency by comparing this micromixing method with other mixing methods such as simple diffusion and the use of a rocking shaker at various working volumes. This method has the potential to be widely utilized in suspension assay technology as an efficient mixing strategy

THE GEOMETRY OF STEAM GENERATOR TUBE AND ITS RELEVANCE TO THE OCCURRENCE OF STRESS CORROSION CRACKING IN OPERATING NUCLEAR POWER PLANTS

INTRODUCTION Occurrences of a stress corrosion cracking in the steam generator tubes of operating nuclear power plants are closely related to the residual stress existing in the local region of a geometric change, that is, expansion transition, u-bend, ding, dent, bulge, etc. Therefore, information on the location, type and quantitative size of a geometric anomaly existing in a tube is a prerequisite to the activity of a non destructive inspection for an alert detection of an earlier crack and the prediction of a further crack evolution In order to verify the performance of the D-probe including the accuracy of the profile measurement and a applicability of the probe to the plant inspection, the quantitatively measured profile data are compared with those from the laser profilometry (measurement resolution of 0.013mm) for the steam generator tube samples of geometric anomalies with various types and sizes, and the relationship between the tube geometry and the evolution of ID and OD side stress corrosion cracking at various tube locations of a steam generator is discussed with the results from the inservice inspections of operating nuclear power plants using a D-probe

Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα

Protein kinase Cα (PKCα) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKCα, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein–protein interaction has limited structural mapping of kinase–substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase–substrate interface. We found that the binding strength of the kinase–substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate–kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKCα for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase–substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases

Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα

Protein kinase Cα (PKCα) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKCα, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein–protein interaction has limited structural mapping of kinase–substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase–substrate interface. We found that the binding strength of the kinase–substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate–kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKCα for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase–substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases

Phlebosclerotic Colitis in a Cirrhotic Patient with Portal Hypertension: The First Case in Korea

Phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the wall of the affected colon due to fibrous degeneration of submucosal layer of colon and fibrotic obstruction of the colono-mesenteric vein, resulting in the disturbance of venous return from the colon. The pathogenic mechanism of this entity remains unknown but chronic liver disease with portal hypertension is maybe thought to be one of the speculated mechanisms. Here we first report the case of surgically confirmed phlebosclerotic colitis, that was in the early stage but showed the aggressive nature, in a 61-yr-old cirrhotic patients with portal hypertension in Korea

Conformational Changes in Two Inter-Helical Loops of Mhp1 Membrane Transporter.

Mhp1 is a bacterial secondary transporter with high-resolution crystal structures available for both the outward- and inward-facing conformations. Through molecular dynamics simulations of the ligand-free Mhp1 as well as analysis of its crystal structures, here we show that two inter-helical loops, respectively located at the extra- and intracellular ends of the "hash motif" in the protein, play important roles in the conformational transition. In the outward- and inward-facing states of the protein, the loops adopt different secondary structures, either wrapped to the end of an alpha-helix, or unwrapped to extended conformations. In equilibrium simulations of 100 ns with Mhp1 in explicit lipids and water, the loop conformations remain largely stable. In targeted molecular dynamics simulations with the protein structure driven from one state to the other, the loops exhibit resistance and only undergo abrupt changes when other parts of the protein already approach the target conformation. Free energy calculations on the isolated loops further confirm that the wrapping/unwrapping transitions are associated with substantial energetic barriers, and consist of multiple sequential steps involving the rotation of certain backbone torsion angles. Furthermore, in simulations with the loops driven from one state to the other, a large part of the protein follows the loops to the target conformation. Taken together, our simulations suggest that changes of the loop secondary structures would be among the slow degrees of freedom in the conformational transition of the entire protein. Incorporation of detailed loop structures into the reaction coordinate, therefore, should improve the convergence and relevance of the resulting conformational free energy

The simulation system.

<p>AdK was initially in the closed conformation in this particular simulation. The AMPbd, LID, and CORE domains of the protein are colored red, yellow, and blue, respectively. K+ and Cl⁻ ions are drawn as blue and red spheres, respectively. The image was rendered using the VMD software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068023#pone.0068023-Humphrey1" target="_blank">[45]</a>.</p

Thickness of the lipid bilayer.

<p>The thickness was calculated as the difference between the average <i>z</i> coordinates (membrane plane normal to the <i>z</i> axis) of the lipid phosphorus atoms in each leaflet. (<b>A</b>) Results for the unbiased simulation with Mhp1 in the OF state. (<b>B</b>) Results for the IF-state simulation.</p