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    Wide-angle x-ray solution scattering

    X-ray solution scattering in both the small-angle (SAXS) and wide-angle (WAXS) regimes is making an increasing impact on our understanding of biomolecular complexes. The accurate calculation of WAXS patterns from atomic coordinates has positioned the approach for rapid growth. WAXS data are sensitive to small structural changes in proteins; useful for calculation of the pair-distribution function at relatively high resolution; provides a means to characterize the breadth of the structural ensemble in solution; and can be used to identify proteins with similar folds. WAXS data are often used to test structural models, identify structural similarities and characterize structural changes. WAXS is highly complementary to crystallography and NMR. It holds great potential for the testing of structural models of proteins; identification of proteins that may exhibit novel folds; characterization of unfolded or natively disordered proteins; and detection of structural changes associated with protein function.
    Current work is aimed at characterizing the structure of intermediates in enzyme catalysis and structural transformations – catching images of proteins while they are at work.
    Makowski, L., D.J. Rodi, S. Mandava, S. Devrapahli, and R.F. Fischetti (2008) Characterization of Protein Fold using Wide Angle X-ray Solution Scattering. J. Mol. Biol. 383, 731-744. PMID: 18786543
    Park, S., J. P. Bardhan, B. Roux, and L. Makowski (2009) Simulated X-Ray Scattering of Protein Solutions Using Explicit-Solvent Molecular Dynamics. J. Chem. Phys. 130, 134114. PMID: 19355724
    Yang, S., S. Park, L. Makowski, and B. Roux (2009) A Rapid Coarse Residue-Based Computational Method for X-Ray Solution Scattering Characterization of Protein Folds and Multiple Conformational States of Large Protein Complexes. Biop. J. 96, 4449–4463. PMID: 19486669
    Bardhan, J.P., S. Park, and L. Makowski (2009) SoftWAXS: A Computational Tool for Modeling Wide-Angle X-ray Solution Scattering from Biomolecules. J.. Appl. Cryst. 42, 932-943
    Makowski, L. (2010) Characterization of Proteins with Wide-angle X-ray Solution Scattering (WAXS). J. Struct. Funct. Genomics. 11, 9-19; PMID:20049539.
    Virtanen, J.J., L. Makowski, T.R. Sosnick and K.F. Freed (2010) Modeling the hydration layer around Proteins: HyPred. Biop. J. 99, 1611-1619. PMID: 20816074.

    Protein Ensembles

    It is becoming increasingly clear that characterization of the protein ensemble – the collection of all conformations of which the protein is capable – will be a critical step in developing a full understanding of the linkage between structure, dynamics and function. X-ray solution scattering in the small angle (SAXS) and wide-angle (WAXS) regimes represents an important new window to exploring the behavior of ensembles. The characteristics of the ensemble express themselves in x-ray solution scattering data in predictable ways. We have collected and analyzed WAXS data on a range of biomolecular systems and demonstrated the modulation of these ensembles by ligand binding; mutation and environmental factors.

    Makowski, L., D. J. Rodi, S. Mandava, D. Minh, . D. Gore and R. F. Fischetti (2008) Molecular crowding inhibits intramolecular breathing motions in proteins. J. Mol. Biol. 375, 529-546. PMID: 18031757
    Makowski, L. D. Gore, S. Mandava, D. Minh, S. Park, D. J. Rodi and R. F. Fischetti (2011) X-ray solution scattering studies of the structural diversity intrinsic to protein ensembles (submitted for publication).
    Yang, S., L. Blachowicz, L. Makowski and B. Roux (2010) Multidomain assembled States of Hck Tyrosine Kinase in Solution. Proc. Natl. Acad. Sci. 107, 15757-62. PMID: 20798061

    see also the News and Views article on this work in Nature: B. Pau and M. Blackledge (2010) Proteins in Dynamic Equilibrium. Nature 468, 1046-1048.
    Neutron Spin Echo Spectroscopy
    Neutron spin-echo spectroscopy is an underutilized probe of protein dynamics sensitive to slow correlated motions (including translational and rotational diffusion and internal modes) on the picosecond to nanosecond time scale. We have used neutron spin-echo (NSE) spectroscopy to study structural fluctuations that occur in hemoglobin (Hb) and myoglobin (Mb) in solution. Using NSE data to very high momentum transfer, q ( ~ 0.62 Å-1), the internal dynamics of these proteins were characterized at the level of the dynamical pair correlation function and self-correlation function in the time range of several picoseconds to a few nanoseconds. Comparison of data from the two homologous proteins collected at different temperatures and protein concentrations was used to assess the contributions to the data made by translational and rotational diffusion and internal modes of motion. The temperature dependence of the decay times can be attributed to changes in viscosity and temperature of the solvent as predicted by the Stokes-Einstein relationship. This is true for contributions from both diffusive and internal modes of motion indicating an intimate relationship between the internal dynamics of the proteins and the viscosity of the solvent. Viscosity change associated with protein concentration can account for changes in diffusion observed at different concentrations, but is apparently not the only factor involved in the changes in internal dynamics observed with change in protein concentration. Data collected at high q indicate that internal modes in Mb are generally faster than in Hb, perhaps due to the greater surface to volume ratio of Mb and the fact that surface groups tend to exhibit faster motion than buried groups. Comparison of data from Hb and Mb at low q indicate an unexpectedly rapid motion of the hemoglobin αβ-dimers relative to one another. Dynamic motion of subunits is increasingly perceived as important to the allosteric behavior of hemoglobin. Our data demonstrate that this motion is highly sensitive to protein concentration, temperature and solvent viscosity, indicating that great care need be exercised in interpreting its effect on protein function.
    Lal, J., P. Fouquet, M. Maccarini, and L. Makowski (2010) Neutron Spin Echo studies of Hemoglobin and Myoglobin: Multiscale Internal Dynamics. J. Mol. Biol. 397, 423-435. PMID: 20096701