Mycobacterium tuberculosis Hsp16.3 nonamers are assembled and re-assembled via trimer and hexamer intermediates

被引:20
作者
Abulimiti, A
Fu, XM
Gu, LC
Feng, XG
Chang, ZY [1 ]
机构
[1] Tsinghua Univ, Dept Biol Sci & Biotechnol, Sch Life Sci, Beijing 100084, Peoples R China
[2] Tsinghua Univ, Dept Biochem & Mol Biol, Sch Med, Beijing 100084, Peoples R China
[3] Educ Minist, Prot Sci Lab, Beijing 100084, Peoples R China
关键词
small heat shock protein16.3; in vitro transcription/translation assembly/re-assembly; subunit exchange; macromolecular crowding;
D O I
10.1016/S0022-2836(03)00018-4
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis proposed to form specific trimer-of-trimers structures, acts as a molecular chaperone in vitro. The assembly and re-assembly mechanisms of this oligomeric protein were studied and compared using in vitro transcription/translation and denaturization/renaturization systems. Analysis using a combination of non-denaturing pore gradient polyacrylamide gel electrophoresis, chemical cross-linking, and size-exclusion chromatography demonstrate that the predominant form of Hsp16.3 produced in the in vitro transcription/translation system is the trimer, which can be further assembled into a nonameric structure via a hexamer intermediate in the presence of purified exogenous Hsp16.3 proteins. Meanwhile, an "inert" Hsp16.3 dimer, which does not seem to participate in nonamer assembly but may be involved in forming other forms of Hsp16.3, was also detected in the in vitro expression system. On the other hand, our current data clearly show that the re-assembly of Hsp16.3 nonamers also occurs via a very similar mechanism, with the formation of trimers and hexamers. The presence of high levels of macromolecular crowding protein agent in the in vitro expression system promoted the formation of the nonamers to a very limited degree, indicating that the assembly of proteins like Hsp16.3 may depend mainly on its own concentration instead of those of the macromolecules in the environment. (C) 2003 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:1013 / 1023
页数:11
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