M R Burgio1, P M Bennett, J F Koretz. 1. Center for Biophysics and Department of Biology, Science Center, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. burgim@rpi.edu
Abstract
PURPOSE: To compare the effects of heat incubation on the structure and function of native alpha-crystallin, urea denatured/renatured alpha-crystallin, and alphaA and alphaB-crystallin homo-polymers purified from bovine lenses. METHODS: Each of the alpha-crystallin samples were incubated for 1 h at temperatures ranging from 35 degrees C to 70 degrees C. After heat incubation structural perturbations in each of the samples were studied using non-denaturing gel electrophoresis, transmission electron microscopy (TEM) and far-UV circular dichroism. The chaperone-like activity of each of the heat-treated samples was measured using the DTT induced insulin aggregation assay. RESULTS: The native alpha-crystallin samples showed secondary structure perturbations, an increase in aggregate size and asymmetry, and an increase in chaperone-like activity after heat incubation above 50 degrees C. The other three sample types showed secondary structure perturbations beginning at lower incubation temperatures, and a progressive decrease in chaperone-like activity with exposure to increasing temperatures. TEM showed all samples formed large asymmetric high molecular weight aggregates after incubation at 65 degrees C. CONCLUSIONS: The urea denaturation/renaturation of alpha-crystallin has been shown to result in the loss of a small amount of alpha-helix, but to have no effect on chaperone-like activity under standard test conditions. The present results indicate this lost alpha-helix may be responsible for the differential effects of heat incubation on the different forms of alpha-crystallin.
PURPOSE: To compare the effects of heat incubation on the structure and function of native alpha-crystallin, urea denatured/renatured alpha-crystallin, and alphaA and alphaB-crystallin homo-polymers purified from bovine lenses. METHODS: Each of the alpha-crystallin samples were incubated for 1 h at temperatures ranging from 35 degrees C to 70 degrees C. After heat incubation structural perturbations in each of the samples were studied using non-denaturing gel electrophoresis, transmission electron microscopy (TEM) and far-UV circular dichroism. The chaperone-like activity of each of the heat-treated samples was measured using the DTT induced insulin aggregation assay. RESULTS: The native alpha-crystallin samples showed secondary structure perturbations, an increase in aggregate size and asymmetry, and an increase in chaperone-like activity after heat incubation above 50 degrees C. The other three sample types showed secondary structure perturbations beginning at lower incubation temperatures, and a progressive decrease in chaperone-like activity with exposure to increasing temperatures. TEM showed all samples formed large asymmetric high molecular weight aggregates after incubation at 65 degrees C. CONCLUSIONS: The urea denaturation/renaturation of alpha-crystallin has been shown to result in the loss of a small amount of alpha-helix, but to have no effect on chaperone-like activity under standard test conditions. The present results indicate this lost alpha-helix may be responsible for the differential effects of heat incubation on the different forms of alpha-crystallin.
Authors: Sarah L Shammas; Christopher A Waudby; Shuyu Wang; Alexander K Buell; Tuomas P J Knowles; Heath Ecroyd; Mark E Welland; John A Carver; Christopher M Dobson; Sarah Meehan Journal: Biophys J Date: 2011-10-05 Impact factor: 4.033