1. 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 1 FLNP, JINR, Dubna, Russia 2 Institute of Biochemical Physics of RAS, Moscow, Russia (the Tasking) 3 Moscow Institute of Physics and Technology, Dolgoprudny, Russia 4 Helmholtz-Zentrum Geesthacht, Zentrum für Material- und Köstenforschung GmbH, Geesthacht, Germany 5 Taras 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 Monomers: αA- and αB-crystallin 20 kDa Oligomer: 20-40 monomers 400-1000 kDa

5. Models of α-crystallin structure 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 Single particle reconstructions of native a-crystallin from Electron microscopy. D.A. Haley et al., J. Mol. Biol. (2000) 298, 261±272 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-crystallin have 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.htmlhttp://www.embl-hamburg.de/biosaxs/software.html.

8. Molecular mass M, volume V and hydration value M = (600±17) kDa → V ah = M·ν = (740±20)·10 3 Å 3 (anhydrous volume) Hydrous volume V h : (Porod invariant) (Fig. 3) Hydration value for α- crystallin: 0.26 g of water per g of protein I·q 2 vs q plot for estimation of Porod volume. The Porod volume was calculated by program PRIMUS [20] where I(0)=2.97 cm -2 (from indirect transformation) N A = 6.022·10 23 (Avogadro constant) c=9 mg/ml (concentration of protein in the sample) (average scattering density of protein) [8] ρ s = -0.548·10 10 cm -2 (scattering density of the solvent) (specific volume) [13]

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

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

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