Abstract
Abstract: :
Purpose: While there is considerable variation in the length and sequence of the C–terminal region of small heat shock protein superfamily members, it usually contains a conserved XYX sequence, where X is a hydrophobic residue and Y is typically a proline. In solved sHSP crystal structures (HSP16.5 and TaHSP16.9), this short sequence forms an isolated beta strand apparently involved in alignment of dimers into larger aggregates. Because it is a common feature of sHSP family members, it is likely that this triad has a similar role in alphaA–crystallin. This study was undertaken to determine the contribution of this consensus triad to the quaternary structure and function of alphaA–crystallin. Methods: A series of site–directed mutants was generated in both wild type alphaA and in an alphaA deletion mutant lacking the N–terminal (residues 1–50). Proteins were expressed in BL21(DE3) E.coli, and purified by ion–exchange and size–exclusion chromatography. Aggregate size was characterized using a Superose HR 10/30 gel exclusion column, thermal transitions by non–denaturing composite gel electrophoresis, and chaperone activity assayed by the inhibition of DTT–induced insulin aggregation. Results:Using the alphaA N–deletion mutant, the hydrophobic triad was changed from IPV to TPT, GPG, IGV, ITV or GGG. All six mutants associated into tetramers, with small amounts of dimer and monomer also present. Chaperone–like activity was reduced but not eliminated in some of the triad mutants, with GGG and ITV the most strongly affected. Similar modifications to wild type alphaA (IPV to ITV, IGV, or GGG) restored aggregate sizes similar but not identical to native, with additional small amounts of tetramer and dimer. Interestingly, equivalent mutants of wild type alphaA did not have reduced chaperone–like activity, but differed considerably in their thermal transition temperatures. Conclusions:The conserved C–terminal triad does not appear to have a strong effect on the steady state aggregation of wild type alphaA–crystallin or its deletion mutant at 25C. It can exert a considerable effect on chaperone–like activity in the absence of the N–terminal, however, and can influence the thermal transition temperature in its presence. These results suggest that the conserved trimer in alphaA is not essential for the formation of tetramers, but contributes to the stability of higher order aggregates.
Keywords: protein structure/function • chaperones • crystallins