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Structure peculiarities of α - crystallin studied by small angle neutron and X-ray scattering

Structure peculiarities of α - crystallin studied by small angle neutron and X-ray scattering. T.N. Murugova 1 , O.I. Ivankov 1,5 , A.I. Kuklin 1,3 , K.O. Muranov 2 , N.B. Poliansky 2 , V.M. Garamus 4 , A.V. Krivandin 2.

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Structure peculiarities of α - crystallin studied by small angle neutron and X-ray scattering

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  1. Structure peculiarities of α-crystallin studied by small angle neutron and X-ray scattering T.N. Murugova1, O.I. Ivankov1,5, A.I. Kuklin1,3, K.O. Muranov2, N.B. Poliansky2, V.M. Garamus4, A.V. Krivandin2 1FLNP, JINR, Dubna, Russia2Institute of Biochemical Physics of RAS, Moscow, Russia (the Tasking)3Moscow Institute of Physics and Technology, Dolgoprudny, Russia4Helmholtz-Zentrum Geesthacht, Zentrum für Material- und Köstenforschung GmbH, Geesthacht, Germany5Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

  2. Function • The main component of the vertebrate eye lens (300 mg/ml) • Provides a proper refractive index of the lens • Chaperon-like activity (forms soluble complexes with destabilized proteins and prevents their unspecific aggregation and uncontrolled denaturation) • Slows down the age-dependent loss of the lens transparency (cataract).

  3. Problem • How does this protein protect the lens? • What is the mechanism of its binding of target proteins ? • What is the role of oligomeric structure and subunit exchange in this mechanism? Knowledge of the quaternary structure of α-crystallin is a missing link in defining a structure-function relationship and the possible role of the a-crystallin subunits in the lens and other organs in normal and abnormal conditions. The quaternary structure of α-crystallin is not known to date.

  4. Structure: oligomeric protein Oligomer: 20-40 monomers 400-1000 kDa Monomers: αA- and αB-crystallin 20 kDa

  5. Models of α-crystallin structure Single particle reconstructions of native a-crystallin from Electron microscopy. D.A. Haley et al., J. Mol. Biol. (2000) 298, 261±272 Aquilina, J. A. and Morris, A. M.: Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin 2010. http://ro.uow.edu.au/scipapers/175 Open micellar configuration B. GROTH-VASSELLI ET AL., Exp. Eye Res. (1995) 61, 249-253 Micellar three-layer model Walsh et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 266 ,pp.20079-20084,1991 3-layer model Tardieu et al., .I. Mol. Biol. (1986) 192,711 -724 A model of αB-crystallin with bound α-lactalbumin . Electron microscopy. Scale bar represents 100 Å. J.Horwitz, Exp. Eye Res. (2003) 76, 145-153

  6. Why Small Angle Scattering • Polydisperse system: variable size and number of monomers • Dynamic system: monomer exchange between oligomers • Efforts to crystallize α-crystallinhave failed • The aggregate is too large for high resolution 2D NMR • Small angle scattering allows to study structure of macromolecules (10-1000 Å) in solution: • Shape • Size • Molecular mass • Volume

  7. Distance distribution function p(r)(indirect Fourier transform) Curves of SANS and SAXS for the same sample of α-crystallin (concentration 9 mg/ml). Distance distribution functions for α-crystallin molecule calculated from SANS and SAXS curves by indirect transform. Program GNOM: http://www.embl-hamburg.de/biosaxs/software.html.

  8. Molecular mass M, volume V and hydration value I·q2 vs q plot for estimation of Porod volume. The Porod volume was calculated by program PRIMUS [20] M = (600±17) kDa → Vah = M·ν = (740±20)·103 Å3 (anhydrous volume) Hydration value for α-crystallin: 0.26 g of water per g of protein where I(0)=2.97 cm-2 (from indirect transformation) NA = 6.022·1023 (Avogadro constant) c=9 mg/ml (concentration of protein in the sample) (average scattering density of protein) [8] ρs = -0.548·1010cm-2 (scattering density of the solvent) (specific volume) [13] Hydrous volume Vh: (Porod invariant) (Fig. 3)

  9. Shape of α-crystallin Model ellipsoidal shell for α-crystallin. Approximation of SANS and SAXS data for α-crystallin by model of 3-axial ellipsoid and 3-axial ellipsoidal shell.

  10. 3D dumm-models Approximation of experimental data by curves for the 3D-damm models. A DUMM-model from SANS C 3-exis ellipsoid from SANS B DUMM-model from SAXS Program DAMMIF : http://www.embl-hamburg.de/biosaxs/software.html.

  11. The summary table of structural parameters of α-crystallin obtained from small angle scattering

  12. Conclusions • The average values of structural parameters of alpha-cryslallin have been obtained • (Volume, size, hydration value) • Ellipsoid-like shape • The presence of internal cavity is available • In case of model ellipsoidal shell the monomers form an unilayer shell

  13. Thank you for your attention!

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