Subunit Exchange of Polydisperse Proteins

The small heat shock protein, -crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the lifetime of an individual. Here, we examine the structural and functional consequences of one particular modification of A-crystallin involving the truncation of 5 C-terminal residues ( A1–168). Using novel mass spectrometry approaches and established biophysical techniques, we show that A1–168 forms oligomeric assemblies with a lower average molecular mass than wild-type A-crystallin ( AWT). Also apparent from the mass spectra of both AWT and A1–168 assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for A1–168. To examine the rate of exchange of subunits between assemblies, we mixed B crystallin with either AWT or A1–168 and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when A1–168 is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both AWT and A1–168. Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian A-crystallin.