Probing Membrane Protein Unfolding with Pulse Proteolysis

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• 作者：Jonathan P. Schlebach¹ ; ² ; Moon-Soo Kim¹ ; ¹ ; Nathan H. Joh³ ; ¹ ; James U. Bowie³ ; Chiwook Park¹ ; ² ; ⁴ ; ^{chiwook@purdue.edu}
• 关键词：bacteriorhodopsin ; membrane proteins ; protein folding ; protein stability ; pulse proteolysis
• 刊名：Journal of Molecular Biology
• 出版年：2011
• 出版时间：4 March 2011
• 年：2011
• 卷：406
• 期：4
• 页码：545-551
• 全文大小：264 K

文摘

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A₅₆₀). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C_m) shows excellent agreement with that determined by A₅₆₀. The C_m values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A₅₆₀. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.