Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis.
Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.
Akhtar
Mohd Waseem
MW
Centre for Cellular and Molecular Biology, Hyderabad-500007, India.
Srinivas
Volety
V
Raman
Bakthisaran
B
Ramakrishna
Tangirala
T
Inobe
Tomonao
T
Maki
Kosuke
K
Arai
Munehito
M
Kuwajima
Kunihiro
K
Rao
Ch Mohan
ChM
D020106:Acrylamide / Q000737:chemistry; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D005976:Glutaral / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000502:physiology*; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014965:X-Rays
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C492165:HSP33 protein, E coli; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D020106:Acrylamide; D012965:Sodium Chloride; D010100:Oxygen; D005976:Glutaral