Investigation of gammaE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering.
alpha-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 alpha-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 (gammaE-crystallin) with alpha-crystallin (alpha(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 alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(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 gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2)O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.
Clarke
M J
MJ
School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cathays, Cardiff, CF24 4LU, UK.
Artero
J B
JB
Moulin
M
M
Callow
P
P
Carver
J A
JA
Griffiths
P C
PC
Haertlein
M
M
Harding
J J
JJ
Meek
K M
KM
Timmins
P
P
Regini
J W
JW
D000818:Animals; D002417:Cattle; D007908:Lens, Crystalline / Q000378:metabolism*; D008970:Molecular Weight; D009502:Neutrons; D012542:Scattering, Radiation; D012984:Software; D012997:Solvents; D013816:Thermodynamics; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*; D038222:gamma-Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D012997:Solvents; D038201:alpha-Crystallins; C107256:crystallin gammaE; D038222:gamma-Crystallins