Analysis of dissociation and unfolding of oligomeric proteins using a flat bed gel electrophoresis at high pressure.
A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.
Paladini
A A
AA
Instituto de Investigaciones en Ingenieria Genetica y Biologia Molecular, Facultad de Ciencias Exactas y Naturales, UBA, Argentina.
Weber
G
G
Erijman
L
L
D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry; D006593:Hexokinase / Q000032:analysis / Q000737:chemistry; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry*; D010762:Phosphorylase a / Q000032:analysis / Q000737:chemistry; D006005:Phosphorylases / Q000032:analysis / Q000737:chemistry; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D014508:Urea
D005656:Fungal Proteins; D046911:Macromolecular Substances; D009842:Oligopeptides; D014508:Urea; D010762:Phosphorylase a; D006005:Phosphorylases; D006593:Hexokinase