Subunit exchange of polydisperse proteins: mass spectrometry reveals consequences of αA-crystallin truncation
The small heat shock protein, α-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 life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of αA-crystallin involving the truncation of 5 C-terminal residues (αA1–168). Using novel mass spectrometry approaches and established biophysical techniques, we show that αA1–168 forms oligomeric assemblies with a lower average molecular mass than wild-type αA-crystallin (αAWT). Also apparent from the mass spectra of both αAWT and αA1–168 assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for αA1–168. To examine the rate of exchange of subunits between assemblies, we mixed αB crystallin with either αAWT or αA1–168 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 αA1–168 is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both αAWT and αA1–168. 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 αA-crystallin.