Probing the roles of the only universally conserved leucine residue (Leu122) in the oligomerization and chaperone-like activity of Mycobacterium tuberculosis small heat shock protein Hsp16.3.
To understand the role of the only universally conserved hydrophobic residue among all the members of the sHsp family, this extremely well conserved Leu122 residue in Hsp16.3 was replaced by valine, alanine, asparigine, or aspartate residues. Only very small amounts of the L122D and L122N mutant Hsp16.3 proteins were expressed in the transformed E. coli; however, both the L122V and L122A were readily expressed. The L122V and L122A mutant proteins had similar oligomeric structures to the wild-type protein at room temperature. Examination of the L122A mutant protein by native pore gradient PAGE and CD spectroscopy, however, revealed a smaller oligomeric size and different secondary structure at 37 degrees C. Both L122V and L122A mutant proteins exhibited significantly lowered chaperone activities. Observations reported here suggest a very important role of this only universally conserved Leu residue in both the formation of specific oligomeric structures and the molecular chaperone activities of Hsp16.3.
Dai
H
H
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China.
Mao
Q
Q
Yang
H
H
Huang
S
S
Chang
Z
Z
D001426:Bacterial Proteins; D001483:Base Sequence; D001704:Biopolymers; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D001704:Biopolymers; D017931:DNA Primers; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria; D018832:Molecular Chaperones; D015335:Molecular Probes; D007930:Leucine