Inter-subunit Cross-linking Suppressed the Dynamic Oligomeric Dissociation of Mycobacterium tuberculosis Hsp16.3 and Reduced Its Chaperone Activity

Xinmiao Fu¹, Wangwang Jiao^1,2, Abuduaini Abulimiti¹, and Zengyi Chang^1,2*

²School of Life Sciences, Peking University, Beijing, P. R. China 100871; fax: 86-10-6275-1526; E-mail: changzy@pku.edu.cn

^* To whom correspondence should be addressed.

Received September 9, 2003

---

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.

---

KEY WORDS: Mycobacterium tuberculosis, Hsp16.3, dissociation, chaperone activity

---