Hsp85 conformational change within the heat shock temperature range.
One of the major mammalian heat shock proteins, hsp85, aggregates extensively when heated in the presence of non-ionic detergents (J Cell. Physiol. 140: 601-607, 1989). The present study used intrinsic fluorescence and susceptibility to tryptic proteolysis to probe hsp85 conformation within the physiological and heat shock temperature ranges. Fluorescence intensity decreased and the emission spectrum was red-shifted (2.5 nm) as hsp85 was heated from 15 degrees to 50 degrees C. Upon heating in the absence of detergent, the red shift, monitored by the ratio of fluorescence emission at 330 nm to that at 350 nm, began at 38 degrees-45 degrees C with a transition midpoint at 45 degrees-50 degrees C, depending on the rate of temperature increase. This transition was masked by 1% n-octyl-O-glucoside - a detergent previously shown to promote aggregation. The spectral changes were not reversible upon cooling to 15 degrees C. Susceptibility to proteolysis in the absence of detergent, measured by the degradation of characteristic large fragments, increased sharply between 40 degrees C and 45 degrees C. These findings suggest that hsp85 undergoes a major conformational change within the range of temperatures known to induce hsp synthesis. This change is consistent with partial unfolding which exposes additional sites to the aqueous environment and influences detergent binding.
Lanks
K W
KW
Department of Pathology, State University of New York-Health Science Center, Brooklyn 11203.
London
E
E
Dong
D L
DL
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006358:Hot Temperature; D008970:Molecular Weight; D010449:Peptide Mapping; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014357:Trypsin
D006360:Heat-Shock Proteins; D014357:Trypsin