Greg L. Hura

Development of an improved four-site water model for biomolecular simulations: TIP4P-Ew

A re-parameterization of the standard TIP4P water model for use with Ewald techniques is introduced, providing an overall global improvement in water properties relative to several popular nonpolarizable and polarizable water potentials. Using high precision simulations, and careful application of standard analytical corrections, we show that the new TIP4P-Ew potential has a density maximum at approximately 1 degrees C, and reproduces experimental bulk-densities and the enthalpy of vaporization, DeltaH(vap), from -37.5 to 127 degrees C at 1 atm with an absolute average error of less than 1%. Structural properties are in very good agreement with x-ray scattering intensities at temperatures between 0 and 77 degrees C and dynamical properties such as self-diffusion coefficient are in excellent agreement with experiment. The parameterization approach used can be easily generalized to rehabilitate any water force field using available experimental data over a range of thermodynamic points.

X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution

Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 Ato 10 Aresolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high- resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein-nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.

What can x-ray scattering tell us about the radial distribution functions of water?

We present an analysis of the Advanced Light Source (ALS) x-ray scattering experiment on pure liquid water at ambient temperature and pressure described in the preceding article. The present study discusses the extraction of radial distribution functions from the x-ray scattering of molecular fluids. It is proposed that the atomic scattering factors used to model water be modified to include the changes in the intramolecular electron distribution caused by chemical bonding effects. Based on this analysis we present a gOO(r) for water consistent with our recent experimental data gathered at the ALS, which differs in some aspects from the gOO(r) reported by other x-ray and neutron scattering experiments. Our gOO(r) exhibits a taller and sharper first peak, and systematic shifts in all peak positions to smaller r. Based on experimental uncertainties, we discuss what features of gOO(r) should be reproduced by classical simulations of nonpolarizable and polarizable water models, as well as ab initio simulation...

A high-quality x-ray scattering experiment on liquid water at ambient conditions

We report a new, high-quality x-ray scattering experiment on pure ambient water using a synchrotron beam line at the Advanced Light Source at Lawrence Berkeley National Laboratory. Several factors contribute to the improved quality of our intensity curves including use of a highly monochromatic source, a well-characterized polarization correction, a Compton scattering correction that includes electron correlation, and more accurate intensities using a modern charge coupled device (CCD) detector. We provide a comprehensive description of the data processing that we have used for correcting systematic errors, and we provide an estimate of our remaining random errors. The resulting error estimates of our data are smaller then the discrepancies between data sets collected in past x-ray experiments. We find that the older x-ray curves support a family of gOO(r)’s that exhibit a smaller first peak (∼2.2), while the current data is better fit with a family of gOO(r)’s with a first peak height of 2.8, and systema...

Hydration dynamics near a model protein surface

The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering (QENS) and molecular dynamics (MD) simulation for both the completely deuterated and completely hydrogenated leucine monomer. The NALMA-water system and the QENS data together provide a unique study for characterizing the dynamics of different hydration layers near a prototypical hydrophobic side chain and the backbone of which it is attached. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational and rotational water dynamics at the highest solute concentrations are found to be highly suppressed as characterized by long residential time and slow diffusion coefficients. The analysis of the more dilute concentration solutions models the first hydration shell with the 2.0 M spectra. We find that for outer layer hydration dynamics that the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis of the first hydration shell water dynamics shows spatially heterogeneous water dynamics, with fast water motions near the hydrophobic side chain, and much slower water motions near the hydrophilic backbone. We discuss the hydration dynamics results of this model protein system in the context of protein function and protein-protein recognition.

Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.

Electron microscopy of gold nanoparticles at atomic resolution

Detailed structure of a gold nanoparticle Adding only a few atoms or changing the capping ligand can dramatically change the structure of individual metal nanoparticles. Azubel et al. used aberration-corrected transmission electron microscopy to derive a three-dimensional reconstruction of water-soluble gold nanoparticles. Small-angle x-ray scattering and other techniques have also corroborated this model. They used this to determine the atomic structure, which compared favorably with density functional theory calculations, without assuming any a priori structural knowledge or the use of model fitting. Science, this issue p. 909 The atomic structure of a 68–gold atom nanoparticle is determined without prior structural knowledge or model fitting. Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102–gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory.

Structure of a designed protein cage that self-assembles into a highly porous cube

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic level agreement with the designed model, while electron microscopy, native mass spectrometry, and small angle x-ray scattering revealed alternate assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will likely require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.

Small-angle scattering and the structure of ambient liquid water

Structural polyamorphism has been promoted as a means for understanding the anomalous thermodynamics and dynamics of water in the experimentally inaccessible supercooled region. In the metastable liquid region, theory has hypothesized the existence of a liquid-liquid critical point from which a dividing line separates two water species of high and low density. A recent small-angle X-ray scattering study has claimed that the two structural species postulated in the supercooled state are seen to exist in bulk water at ambient conditions. We analyze new small-angle X-ray scattering data on ambient liquid water taken at third generation synchrotron sources, and large 32,000 water molecule simulations using the TIP4P-Ew model of water, to show that the small-angle region measures standard number density fluctuations consistent with water’s isothermal compressibility temperature trends. Our study shows that there is no support or need for heterogeneities in water structure at room temperature to explain the small-angle scattering data, as it is consistent with a unimodal density of the tetrahedral liquid at ambient conditions.

Three-dimensional structure of the KChIP1-Kv4.3 T1 complex reveals a cross-shaped octamer

Brain IA and cardiac Ito currents arise from complexes containing Kv4 voltage-gated potassium channels and cytoplasmic calcium-sensor proteins (KChIPs). Here, we present X-ray crystallographic and small-angle X-ray scattering data that show that the KChIP1–Kv4.3 N-terminal cytoplasmic domain complex is a cross-shaped octamer bearing two principal interaction sites. Site 1 comprises interactions between a unique Kv4 channel N-terminal hydrophobic segment and a hydrophobic pocket formed by displacement of the KChIP H10 helix. Site 2 comprises interactions between a T1 assembly domain loop and the KChIP H2 helix. Functional and biochemical studies indicate that site 1 influences channel trafficking, whereas site 2 affects channel gating, and that calcium binding is intimately linked to KChIP folding and complex formation. Together, the data resolve how Kv4 channels and KChIPs interact and provide a framework for understanding how KChIPs modulate Kv4 function.