title: Probing membrane protein unfolding with pulse proteolysis.
abstract: 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 (A(560)). 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 A(560). The C(m) values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A(560). 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.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Schlebach
authors.forename: Moon-Soo
authors.initials: MS
authors.lastname: Kim
authors.forename: Nathan H
authors.initials: NH
authors.lastname: Joh
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
authors.forename: Chiwook
authors.initials: C
authors.lastname: Park
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D019617:Halobacterium salinarum / Q000737:chemistry; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D020860:Subtilisin / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; D020860:Subtilisin