Mycobacterium tuberculosis Hsp16.3 nonamers are assembled and re-assembled via trimer and hexamer intermediates.
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.
Abulimiti
Abuduaini
A
Department of Biological Science and Biotechnology, School of Life Science, Tsinghua University, Beijing 100084, People's Republic of China.
Fu
Xinmiao
X
Gu
Liangcai
L
Feng
Xiuguang
X
Chang
Zengyi
Z
D001426:Bacterial Proteins; D002474:Cell-Free System; D018833:Chaperonins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D014158:Transcription, Genetic; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; C431133:Hsp16.3 protein, Mycobacterium tuberculosis; D046911:Macromolecular Substances; D014357:Trypsin; D018833:Chaperonins