posters 5th Asia-Pacific NMR Symposium 2013

Characterisation of an 100 kDa multidomain molecular chaperone by SAXS, ESR and NMR (#137)

Chih Ting Huang 1 , Po-Min Shih , Iren Wang , Kuang-Yu Chang , Shang-Te Danny Hsu
  1. Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

Trigger factor (TF) is a conserved multidomain molecular chaperone in bacteria that interacts with newly synthesized polypeptide chains as they emerge from the ribosome. In contrast with other molecular chaperones, e.g., DnaK/DnaJ and GroEL/GroES, the chaperone activity of TF is independent of ATP hydrolysis. Hence understanding its intrinsic dynamics and domain architecture is essential for understanding the underlying mechanism of its chaperone activity. Using size-exclusion chromatography coupled multiangle light scattering (SEC-MALS) and analytical ultracentrifugation (AUC), we first investigate the monomer-dimer equilibrium of TF and the contributions from its three domains, namely the ribosome-binding domain (RBD), substrate binding domain (SBD) and peptidyl prolyl cis-trans isomerase domain (PPI). We next combine small angle X-ray scattering (SAXS), electron spin resonance (ESR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to investigate the domain assembly of TF in its homodimeric state in solution at an apparent molecular weight of ca. 100 kDa. The combined use of ESR and NMR using site-specifically spin labelled TF variants is instrumental for extracting long-range intermolecular contacts and the population distributions up to 70 Å. Our results show that the dimeric TF exists in an ensemble of conformations that are different from the reported crystal structures, that there exist at least three distinct populations and that the three domains display a broad range of internal dynamics which may be associated with its chaperone activity.