NH2-terminal stabilization of small heat shock protein structure: a comparison of two NH2-terminal deletion mutants of alphaA-crystallin.
OBJECTIVE
To assess the role of the NH2-terminal in alphaA-crystallin folding and chaperone-like activity.


METHODS
Two NH2-terminal deletion mutants of alphaA-crystallin were generated by standard mutagenesis methods, one with and one without a leader sequence in place of the first 50 residues. Aggregate size of each before and after thermal stress was assessed by FPLC, and chaperone-like activity was assessed using DTT-induced insulin denaturation.


RESULTS
Both mutants assemble primarily into tetramers, and both exhibit similar levels of chaperone-like activity, but are less protective than recombinant alphaA-crystallin. After a cycle of heat stress to 70 degrees C, tetramers of the mutant without the leader sequence dissociate into dimers and monomers and show severely reduced chaperone-like activity. In contrast, the mutant with the leader sequence retains its tetrameric form and its chaperone-like activity.


CONCLUSIONS
The NH2-terminal region is an important determinant of alpha-crystallin aggregate size, but is not required for folding of the alpha-crystallin domain, since the aggregate size and chaperone-like activity of the two mutants at room temperature are essentially the same. The leader sequence appears to increase the thermal stability of the alpha-crystallin domain and/or to contribute to the reformation of the active form after cooling, suggesting that the native NH2-terminal also plays a role in alpha-crystallin's resistance to environmental stress.
Yang
Chaoxing
C
Biochemistry and Biophysics Program, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA.
Salerno
John C
JC
Koretz
Jane F
JF
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017384:Sequence Deletion; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics
D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D038202:alpha-Crystallin A Chain