Evidence for specific subunit distribution and interactions in the quaternary structure of alpha-crystallin.
The quaternary structure of alpha-crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision-induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of alpha-crystallin. For total alpha-crystallin isolated directly from fetal calf lens using size-based chromatography, the alphaB-crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the alphaA-crystallin subunits. Furthermore, upon mixing molar equivalents of purified alphaA- and alphaB-crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the alphaA- and alphaB-crystallin homo-oligomers was comparable, indicating that strength of the alphaA:alphaA, and alphaB:alphaB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of alphaB-crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C-terminus of alphaA-crystallin was more susceptible to proteolytic attack in homo-oligomers than that of alphaB-crystallin. As subunit exchange proceeded, proteolysis of the alphaA-crystallin C-terminus increased, indicating that in the hetero-oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the alpha-crystallin domains and C-terminal extensions of subunits within the alpha-crystallin assembly. In particular, we propose that the palindromic IPI motif of alphaB-crystallin gives rise to two orientations of the C-terminus.
School of Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522, Australia.
Amie M
AM
Morris
J Andrew
JA
Aquilina
D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D005333:Fetus; D007908:Lens, Crystalline / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D038201:alpha-Crystallins / Q000737:chemistry* / Q000378:metabolism
D021122:Protein Subunits; D038201:alpha-Crystallins