Physical-chemical characterization of small heat shock proteins 12.2/12.3 from Caenorhabditis elegans:evaluation of chaperone activity in the smallest heat shock proteins

• Main Author: 謝孟穎
• Other Authors: Shyh-Horng Chiou
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/87675793609313524610

碩士 === 國立臺灣大學 === 生化科學研究所 === 92 === Small heat shock proteins (sHSPs) represent an abundant and ubiquitous family of stress proteins. The proteins range in size from 12 to 45 kDa and are found as large complexes of 200-800 kDa. sHSPs show little sequence homology. The homology is only restricted to the evolutionarily conserved region termed the ‘α-crystallin domain’. The N- and C- extensional sequences outside this common domain vary in length and amino acid composition. The Caenorhabditis elegans genome project was completed in 1998, making it the first multicellular organism for which the genome has been sequenced. As expected from evolutionary relationships, there were more protein similiarities found between C. elegans and eukaryotic organisms than that between cross-species pairwise comparison with prokaryotes. Here, we use C. elegans as a model to investigate the structure and function of sHSPs. This study is to characterize and compare the physical-chemical properties and chaperone function of sHSP12.2 and sHSP12.3 from C. elegans. According to the ultracentrifugation analysis and cross-linking assay, two proteins both form a tetrameric complex. Besides, sHSP12.3 (Tm＞90℃) is more thermostable than sHSP12.2 (Tm＝62℃). Estimation of secondary structure based on circular dichroism, sHSP12.2 possesses 25% β-strand, while sHSP12.3 has about 40% β-strand composition. Their secondary structures were converted to unordered form as the temperature increased to 80℃ from 25℃. This observation is accompanied by the decrease of the intrinsic and extrinsic fluorescence of the proteins, indicating that accessibility of tryptophan residues to solvent is enhanced. These changes may contribute to the total loss of their weak chaperone activity, and the aggregation of denatured substrates. The result is different from the previous study indicating that preheated sHSPs possess better chaperone activities. Furthermore, sHSP12.2 and sHSP12.3 in C. elegans were identified by 2D-eletrophoresis coupled with MALDI - TOF mass spectrometry from cultures under heat stress. Detailed biological roles of these sHSPs in vivo remain to be studied in the future.