N- and C-Terminal Motifs in Human B Crystallin Play an Important Role in the Recognition, Selection, and Solubilization of Substrate

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• 作者：Joy G. Ghosh ; Ananth K. Shenoy ; Jr. ; John I. Clark
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：November 21, 2006
• 年：2006
• 卷：45
• 期：46
• 页码：13847 - 13854
• 全文大小：458K
• 年卷期：v.45,no.46(November 21, 2006)
• ISSN：1520-4995

文摘

The functions of the interactive sequences in human G SRC="/images/gifchars/alpha.gif" BORDER=0>B crystallin that are involved in chaperoneactivity and complex assembly of small heat shock proteins need to be characterized to understand themechanisms of action on unfolding and misfolding proteins. Protein pin arrays identified the hydrophobicN-terminal sequence (₄₁STSLSPFYLRPPSFLRAP₅₈) and the polar C-terminal sequence (₁₅₅PERTIPITREE₁₆₅) as interactive domains in human ges/gifchars/alpha.gif" BORDER=0>B crystallin, which were then deleted to evaluate theirimportance in complex assembly and chaperone activity. Size exclusion chromatography determined thatthe complexes formed by the deletion mutants, ges/gifchars/Delta.gif" BORDER=0 >41-58 and ges/gifchars/Delta.gif" BORDER=0 >155-165, were larger and more polydispersethan the wild-type (wt) ges/gifchars/alpha.gif" BORDER=0>B crystallin complex. In chaperone assays, the ges/gifchars/Delta.gif" BORDER=0 >41-58 mutant was as effectiveas wt ges/gifchars/alpha.gif" BORDER=0>B crystallin in protecting partially unfolded ges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">_L crystallin and alcohol dehydrogenase (ADH) andsignificantly less effective than wt ges/gifchars/alpha.gif" BORDER=0>B crystallin in protecting unfolded citrate synthase (CS) fromaggregation. Chaperone activity did not correlate with complex size but corresponded with the amount ofsubstrate protein unfolding. The results confirmed the importance of N-terminal residues 41-58 in selectiveinteractions with completely unfolded substrates. Poor solubility and limited or no chaperone activity forthe three substrates characterized the ges/gifchars/Delta.gif" BORDER=0 >155-165 deletion mutant, which demonstrated the importance ofC-terminal residues 155-165 in maintaining the solubility of unfolded substrates in a manner independentof the amount of substrate protein unfolding. The results presented in this report established that interactivedomains in the N- and C-termini of human ges/gifchars/alpha.gif" BORDER=0>B crystallin are important for the recognition, selection, andsolubility of unfolding substrate proteins.