Hydration Water Dynamics and Instigation of Protein Structural Relaxation

The molecular mechanism of the solvent motion that is required to instigate the protein structural relaxation above a critical hydration level or transition temperature has yet to be determined. In this work we use quasi-elastic neutron scattering (QENS) and molecular dynamics simulation to investigate hydration water dynamics near a greatly simplified protein surface. We consider the hydration water dynamics near the completely deuterated N-acetyl-leucine-methylamide (NALMA) solute, a hydrophobic amino acid side chain attached to a polar blocked polypeptide backbone, as a function of concentration between 0.5M-2.0M, under ambient conditions. In this Communication, we focus our results of hydration dynamics near a model protein surface on the issue of how enzymatic activity is restored once a critical hydration level is reached, and provide a hypothesis for the molecular mechanism of the solvent motion that is required to trigger protein structural relaxation when above the hydration transition.Comment: 2 pages, 2 figures, Communicatio

Software for the high-throughput collection of SAXS data using an enhanced Blu-Ice/DCS control system

The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide one potential route for other SAXS beamlines in need of robust and efficient beamline control software

Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source.

The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world's mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B(4)C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources

Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from \u3ci\u3eEscherichia coli\u3c/i\u3e

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon. Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization. Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs. Significance: The first three-dimensional structural information for any trifunctional PutA is reported

Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from \u3ci\u3eEscherichia coli\u3c/i\u3e

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon. Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization. Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs. Significance: The first three-dimensional structural information for any trifunctional PutA is reported

Title: Water Structure as a Function of Temperature from X-ray Scattering Experiments and Ab Initio Molecular Dynamics

We present high-quality x-ray scattering experiments on pure water taken over a temperature range of 2°C to 77°C using a synchrotron beam line at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. The ALS x-ray scattering intensities are qualitatively different in trend of maximum intensity over this temperature range compared to older x-ray experiments. While the common procedure is to report both the intensity curve and radial distribution function(s), the proper extraction of the real-space pair correlation functions from the experimental scattering is very difficult due to uncertainty introduced in the experimental corrections, the proper weighting of OO, OH, and HH contributions, and numerical problems of Fourier transforming truncated data in Q-space. Instead we consider the direct calculation of xray scattering spectra using electron densities derived from density functional theory based on real-space configurations generated with classical water models. The simulation of the experimental intensity is therefore definitive for determining radial distribution functions over a smaller Q-range. We find that the TIP4P, TIP5P and polarizable TIP4P-Pol2 water models, with DFT-LDA densities, show very good agreement with the experimental intensities, and TIP4P-Pol2 in particular shows quantitative agreement over the full temperature range. The resulting radial distribution functions from TIP4P-Pol2 provide the current best benchmarks for real-space water structure over the biologically relevant temperature range studied here. 1 Introduction Liquid water structure is characterized by x-ray (or neutron) diffraction that measures experimental intensities as a function of momentum transfer, Q=4πsin(θ/2)/λ, where λ is the wavelength and θ is the scattering angle with respect to the incident beam. The most recent x-ray data taken at ambient conditions at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory exhibited significant differences when compared to the scattering curves of past x-ray experiments The intensity is the true experimental observable in which error-bars are well-defined. However, it is typical practice for water scattering experiments to also report radial distributions in addition to the intensity profile, primarily because it is more convenient and practical to consider water structure in terms of real-space distribution functions (1) ( In this study we consider the direct calculation of x-ray scattering spectra using ab-initio density functional theory with the LDA functional over the temperature range studied by experiment. The generation of the real-space "snapshots" could come from either a first 3 principles molecular dynamics calculation Experimental Methods Experimental setup. The data collection was performed at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory on beam line 7.3.3. Doubly distilled and degassed water was used in each experiment, and several data sets with independent fillings of the sample holder were collected at each temperature (2°C, 11°C, 22°C, 33°C, 44°C, 55°C, 66°C, and 77°C). A silicon monochrometer was used to produce an x-ray beam with spot size on the order of 100µm, with 99% of the energy at 12.800 ± 0.001 keV or a wavelength of 0.9686Å. This wavelength provided the best compromise between sufficient flux and maximizing our 4 accessible Q-range. Data sets were collected with a flat slab water sample using a transmisson geometry, with the sample tilted with respect to the incoming x-ray beam by an angle of 30 o . A Bruker Charge Coupled Device (CCD) area detector, mounted on a Huber diffractometer and with dimensions of 9.6cm x 9.6cm collected the diffracted x-rays. In order to realize the full range of 0.1Å -1 < Q <11.1Å -1 provided in this study, the data were collected with the detector in three different positions which were pieced together for the final result. The sample to detector positions were determined to 100µm precision while the angle between the xray beam and the normal to detector face were characterized on the order of mrads. The geometries at each detector position were determined by collecting PbS powder patterns with the powder placed within the sample holder and fitting the resulting sharp Bragg rings to determine all the geometric parameters. A more detailed description of this procedure can be found in previous work Experimental corrections. The collected raw intensity data was transformed to a circularly integrated scattering cross-section on a per electron scale versus Q. The following divides the corrections into two parts. The first deals with corrections that must be applied generally to all detector positions such as absorption, geometric corrections, and polarization of the radiation. The second set of corrections deals with overlapping data from different panels and thus the contribution of background and sample holder scattering. Corrections to the intensities due to absorption by air, water, and window material are given by the form where I o is the intensity if there were no absorption in the sample, I is the measured intensity, t is the thickness, τ is the angle between the plane of the sample and the incident x-ray beam, and ν has the form ν=cosθsinτ+sinθcosφcosτ. The absorption coefficients, µ ρ , can be obtained from http://www-cxro.lbl.gov/optical_constants using a tabulated format based on 6 When the incident radiation is plane-polarized as in our experiment, the in-plane and outof-plane polarization is treated separately, and the measured intensities must be rescaled by the factor ( The data were also corrected for the 1/r 2 fall off of intensity, and correction for pixel orientation with respect to the incident radiation. We discovered that detector manufacturers attempt to provide a correction of this sort in their bundled software for data read-out. Corrections to the image collected by the CCD includes distortions introduced by the fiber optic tapers connecting the plate to the chip, a dark current correction that takes into account photoelectrons ejected by thermal motions in the CCDs and any low-level background radiation in the experimental hutch, and finally a flat field correction that provides a correction for variations in pixel sensitivity. This correction involves a calibration measurement that collects a reference image intensity using either a radioactive source or scattering from a fluorescent material at a set distance from the detector. The image is used to generate a scale factor for each pixel such that, when applied to the flat field image, each pixel measures the same intensity. The set distance with which this correction is calibrated varies among manufacturers. If the calibration experiment involves a relatively small source to detector distance, then the geometric corrections (1/r 2 fall off, pixel orientation) are implicitly taken into account by the manufacturer in the flat field correction. This is the case for the Bruker detector, so we need to "uncorrect" the collected image for the manufacturers geometry, and reapply the correct geometric corrections for our experiment. This is an insidious problem since these detector corrections are applied 7 before the "raw image" is available to the experimentalist, and the correction is not documented in the manufacturer literature. We next turn to the procedure for the subtraction of background that is required in order to match segments of the intensity over the full range of Q in this experiment

From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2