The Mechanism of Fast-Gate Opening in ClC-0

ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237–250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent

The Role of a Conserved Lysine in Chloride- and Voltage-dependent ClC-0 Fast Gating

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening

A proximal femoral implant preserves physiological bone deformation: a biomechanical investigation in cadaveric bones

The aim of this study was to compare the perturbances in bone deformation patterns of the proximal femur due to a conventional cemented femoral stem and a novel uncemented implant designed on the principles of osseointegration. Five matched pairs of fresh frozen human femora were mechanically tested. Bone deformation patterns, measured with a video digitizing system under 1.5 kN joint force, showed that the cemented Spectron femoral implant caused significant alterations to the proximal femoral deformation pattern, whereas the Gothenburg osseointegrated titanium femoral implant did not significantly alter the bone behaviour (p < 0.05). Vertical micromotions measured under 1 kN after 1000 cycles were within the threshold of movement tolerable for bone ingrowth (21 microm for the Gothenburg system and 26 microm for the cemented implant).Published versio

CRANKITE: a fast polypeptide backbone conformation sampler

Background: CRANKITE is a suite of programs for simulating backbone conformations of polypeptides and proteins. The core of the suite is an efficient Metropolis Monte Carlo sampler of backbone conformations in continuous three-dimensional space in atomic details. Methods: In contrast to other programs relying on local Metropolis moves in the space of dihedral angles, our sampler utilizes local crankshaft rotations of rigid peptide bonds in Cartesian space. Results: The sampler allows fast simulation and analysis of secondary structure formation and conformational changes for proteins of average length

Appearance of breast cysts in planar geometry photoacoustic mammography using 1064-nm excitation

In the search for improved imaging modalities for detection and diagnosis of breast cancer, a high negative prediction value is also important. Photoacoustic (optoacoustic) imaging is a relatively new technique that has high potential for visualizing breast malignancies, but little is known about the photoacoustic appearance of benign lesions. In this work, we investigate the visibility of benign breast cysts in forward-mode photoacoustic mammography using 1064-nm light, as currently applied in the Twente photoacoustic mammoscope. Results from (Monte Carlo and k-wave) simulations and phantom measurements were used to interpret results from patient measurements. There was a strong agreement among the results from simulations, phantom, and patient measurements. Depending on the absorption contrast between cyst and breast tissue, cysts were visible as either one or two confined high-contrast areas representing the front and the back of the cyst, respectively. This edge enhancement is most likely the consequence of the local sudden change in the absorbed energy density and Grüneisen coefficients. Although the current forward-mode single-wavelength photoacoustic mammoscope cannot always unambiguously discriminate cysts from malignancies, this study reveals specific features of cysts compared to malignancies, which can be exploited for discrimination of the two abnormalities in future modifications of the image

Engineering of Cyclodextrin Product Specificity and pH Optima of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 β-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Å resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced α-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (β- and γ-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of α-CD. Glu258 is involved in catalysis in CGTases as well as α-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

Most Common Pitfalls Within Creation of Project Proposals for EU Funding

In this funding cycle, method of work of EU finan¬cing sources, along with same success rate, remained very similar to previous ones. So far gathered evaluators` experience, demonstrates the presence of same pitfalls in this financial round, as in previous ones. This paper addresses most com¬mon and obvious pitfalls associated with the pro¬cess of project proposal creation, usual reasons for their occurrence and with some recommendations to overcome them