Investigation of γE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering M.J. Clarke a , J.B. Artero c , M. Moulin d , P. Callow e , J.A. Carver f , P.C. Griffiths b , M. Haertlein d , J.J. Harding g , K.M. Meek a , P. Timmins e , J.W. Regini a, ⁎ a School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cathays, Cardiff, CF24 4LU, UK b School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, UK c EPSAM/ISTM Research Institutes, Keele University, Staffordshire ST5 5BG, England d Institut Laue-Langevin, ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, Rue Jules Horowitz, BP 156, 38042 Grenoble Cedex 09, France e Institut Laue-Langevin, Rue Jules Horowitz, BP 156, 38042 Grenoble Cedex 09, France f School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005, Australia g Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK abstract article info Article history: Received 11 August 2009 Received in revised form 11 November 2009 Accepted 1 December 2009 Available online 11 December 2009 Keywords: Crystallin Chaperone Neutron scattering X-ray scattering Heat shock protein α-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between α-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (γE-crystallin) with α- crystallin (α H ) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the α H in D 2 O incubated at 65 °C increased from 69 ± 3 to 81 ± 5 Å over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated γE-crystallin in H 2 O buffer (γE D /H 2 O) and hydrogenous γE-crystallin in D 2 O buffer (γE H /D 2 O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate γE:α H complexes. The evolution of the aggregation size/shape as an indicator of α H chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 °C) for binary mixtures of α H , γE H , and γE D . It was found that Rg(α H :γE D /D 2 O) N Rg(α H :γE H /D 2 O) N Rg(α H /D 2 O) and that Rg(α H :γE H / D 2 O) ≈Rg(α H /D 2 O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that γE-crystallin binds in a central cavity of the α-crystallin oligomer, during chaperone action. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. 1. Introduction α-Crystallin is one of the major constituent proteins within the crystalline lens of many vertebrates and can approach 50% of the total dry weight of the lens [1,2]. In mammals such as cows, α-crystallin exists as a heterogeneous polydisperse globular protein with an average molecular mass of 700 kDa [3], with a range from 300 kDa to 1.5 MDa depending on the local cellular environment [4]. α-Crystallin consists of two closely related subtypes, αA- and αB-crystallin, with a 57% sequence homology [5]. Both have a subunit mass of approxi- mately 20 kDa [3]. Of these two subtypes, αB-crystallin is widely expressed throughout the major tissues of the body, with αA- crystallin generally being considered lenticular. Within the bovine lens, the subunits assemble in a non-covalent manner in a 3:1 ratio of αA to αB [6]. The protein concentration within the crystallin lens is extremely high, which contributes to the maintenance of the short- range order within the lens cytoplasm and maintains the refractive index necessary for the refraction of light onto the retina. The presence of liquid-like, short-range order ensures transparency within the visible light region of the electromagnetic spectrum [7]. One of the main functions of α-crystallin is to act as a molecular chaperone and prevent large-scale protein aggregation under condi- tions of cellular stress such as those arising from elevated temperature or disease [8]. In vitro, α-crystallin prevents thermal aggregation and precipitation of target proteins [9]. Unlike some other chaperone proteins such as GroEL [10], α-crystallin does not hydrolyse ATP and does not refold denatured proteins. The polydisperse and dynamic nature of α-crystallin has contributed to an inability to crystallise the protein for structural analysis by X-ray crystallography. Therefore, a definitive high-resolution structure has remained elusive. Biochimica et Biophysica Acta 1800 (2010) 392–397 ⁎ Corresponding author. Fax: +44 2920 87 4859. E-mail address: ReginiJ@cardiff.ac.uk (J.W. Regini). 0304-4165/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.12.001 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen