On the mechanism of chaperone activity of the small heat-shock protein of Methanococcus jannaschii.
The small heat-shock protein (sHSP) from Methanococcus jannaschii (Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of approximately 120 A and an internal diameter of approximately 65 A with six square "windows" of approximately 17 A across and eight triangular windows of approximately 30 A across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified single-chain monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80 degrees C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80 degrees C, but substantial conformational changes of the substrate are required for complex formation at 80 degrees C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by "preassembly condensation" of multiple monomers before final assembly of the sphere.
Kim
Rosalind
R
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Lai
Luhua
L
Lee
Hi-Hong
HH
Cheong
Gang-Won
GW
Kim
Kyeong Kyu
KK
Wu
Zheng
Z
Yokota
Hisao
H
Marqusee
Susan
S
Kim
Sung-Hou
SH
D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002942:Circular Dichroism; D019286:Endopeptidase K / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008956:Models, Chemical; D010940:Plant Proteins / Q000737:chemistry; D011489:Protein Denaturation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D019843:Archaeal Proteins; D001426:Bacterial Proteins; C113795:HSP16.5 protein, Methanococcus jannaschii; D006360:Heat-Shock Proteins; D010940:Plant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii; D019286:Endopeptidase K