Association behaviour of human betaB1-crystallin and its truncated forms

Abstract

betaB1-crystallin plays an important role in the assembly of betaH-crystallin yet is known to be subject to N-terminal sequence truncations during human lens development and ageing. Here we have over-expressed human betaB1-crystallin, and various truncated forms in Escherichia coli and used mass spectrometry to monitor the monomer molecular weight. Gel permeation chromatography and laser light scattering have been used to estimate the assembly size of the various polypeptides as a function of protein concentration. The full-length betaB1-crystallin behaves as a dimer, like recombinant human betaB2-crystallin, but undergoes further self-association at high protein concentrations, unlike the betaB2-crystallin. Major truncations from the N-terminal extension lead to anomalous behaviour on gel permeation chromatography indicative of altered interactions with the column matrix, whereas light scattering indicated dimers at low protein concentration that self-associate as a function of protein concentration. Loss of 41 residues from the N-terminus, equivalent to an in vivo truncation site, resulted in temperature-dependent phase separation behaviour of the shortened betaB1-crystallin. Good crystals have been grown of a truncated version of human betaB1-crystallin using an in vitro cleavage protocol.