RIS ID

33784

Publication Details

Morris, A. M. & Aquilina, J. Andrew. (2010). Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin . Proteins: Structure, Function, and Bioinformatics, 78 (11), 2546-2553.

Abstract

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 alpha B-crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the alpha A-crystallin subunits. Furthermore, upon mixing molar equivalents of purified alpha A- and alpha B-crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the alpha A- and alpha B-crystallin homo-oligomers was comparable, indicating that strength of the alpha A:alpha A, and alpha B:alpha B 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 alpha B-crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C-terminus of alpha A-crystallin was more susceptible to proteolytic attack in homo-oligomers than that of aB-crystallin. As subunit exchange proceeded, proteolysis of the alpha A-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 alpha B-crystallin gives rise to two orientations of the C-terminus.

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Link to publisher version (DOI)

http://dx.doi.org/10.1002/prot.22766