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Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase
Published on: February 12, 2019
Probing membrane protein unfolding with pulse proteolysis.
Jonathan P Schlebach1, Moon-Soo Kim, Nathan H Joh
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA.
Journal of Molecular Biology
|January 4, 2011
Summary
Pulse proteolysis offers a new method for studying membrane protein unfolding. This technique, applied to bacteriorhodopsin, accurately quantifies unfolding transitions, potentially enabling in situ analysis without protein purification.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Investigating membrane protein folding and unfolding is challenging due to technical limitations.
- Existing methods for monitoring protein unfolding often require purified proteins or large sample amounts.
Purpose of the Study:
- To evaluate pulse proteolysis as a novel tool for probing membrane protein unfolding.
- To assess the quantitative accuracy of pulse proteolysis for membrane proteins using bacteriorhodopsin as a model.
Main Methods:
- Pulse proteolysis was employed to monitor the unfolding of bacteriorhodopsin in response to varying concentrations of sodium dodecyl sulfate (SDS).
- Proteolytic susceptibility changes were assessed using electrophoresis to quantify intact protein.
- Results were compared with traditional spectrophotometric measurements (absorbance at 560 nm).
Main Results:
- Denaturation of bacteriorhodopsin by SDS significantly increased its susceptibility to subtilisin proteolysis.
- Pulse proteolysis revealed a cooperative unfolding transition for bacteriorhodopsin in SDS.
- The transition midpoint (Cm) determined by pulse proteolysis closely matched spectrophotometric measurements for both wild-type and mutant bacteriorhodopsins.
Conclusions:
- Pulse proteolysis is a validated quantitative tool for studying membrane protein unfolding.
- This method is sensitive and requires minimal protein, potentially allowing in situ analysis with techniques like Western blotting without prior purification or overexpression.
