Inter-subunit cross-linking suppressed the dynamic oligomeric dissociation of Mycobacterium tuberculosis Hsp16.3 and reduced its chaperone activity.
Small heat shock proteins (sHsps) usually exist as dynamic oligomers and oligomeric dissociation was believed to be a prerequisite for their chaperone activities. The truth of this hypothesis was verified in our present study on Hsp16.3, one member of sHsps from Mycobacterium tuberculosis, mainly by utilizing chemical cross-linking. Analysis using size exclusion chromatography demonstrated that the heat-induced oligomeric dissociation of Hsp16.3 was severely blocked due to highly efficient inter-subunit cross-linkages generated by chemical cross-linking, as well as its chaperone activity being reduced. Further analysis by non-denaturing pore gradient polyacrylamide gel electrophoresis and fluorescence spectrometry revealed that the dynamic oligomeric dissociation/reassociation process of Hsp16.3 at room temperature was suppressed by inter-subunit cross-linkages, accompanied by significantly decreased exposure of hydrophobic surfaces that are usually hidden in oligomers. These findings supported the hypothesis that substrate-binding sites of sHsps are exposed presumably by dissociation of larger oligomers into smaller active oligomers, and therefore such a dissociation process could be adjusted to modulate chaperone activities.
Fu
Xinmiao
X
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, P. R. China.
Jiao
Wangwang
W
Abulimiti
Abuduaini
A
Chang
Zengyi
Z
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018833:Chaperonins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D015180:Electrophoresis, Gel, Two-Dimensional; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; C431133:Hsp16.3 protein, Mycobacterium tuberculosis; D018833:Chaperonins