2College of Life Science, Peking University, Beijing 100871, China
3Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
* To whom correspondence should be addressed.
Received March 23, 2005; Revision received May 25, 2005
Small heat shock proteins (sHSPs) are highly divergent in primary sequences, with short conserved motifs found in various subfamilies. Here a Pro-Gly doublet was found to be conserved in most non-animal sHSPs by sequence analysis of a total of 344 unique sHSPs (covering the subfamilies: bacterial class A, bacterial class B, archae, fungi, plant, and animal) placed in data banks. In contrast, the residues corresponding to this Pro-Gly doublet in most of animal sHSPs are often charged. Site-directed mutagenesis studies of Mycobacterium tuberculosis Hsp16.3 replacing the Gly (at position 59) residue by Cys or Trp demonstrate that this Gly is likely involved in subunit interactions, which is consistent with that in Methanococcus jannaschii Hsp16.5 and wheat Hsp16.9. Our data suggest that this Pro-Gly doublet in Hsp16.3 is not directly involved in binding of denatured substrate proteins, whereas the corresponding charged residues in bovine alpha-crystallin were instead proposed before to be involved in substrate binding. These observations indicate that the highly conserved Pro-Gly doublet is critical to discriminate between non-animal and animal sHSPs.
KEY WORDS: chaperone, small heat shock protein, evolution, cysteine modification, tryptophan fluorescence, Hsp16.3