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Probing membrane protein unfolding with pulse proteolysis - PubMed

  • ️Sat Jan 01 2011

Review

Probing membrane protein unfolding with pulse proteolysis

Jonathan P Schlebach et al. J Mol Biol. 2011.

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.

Copyright © 2011 Elsevier Ltd. All rights reserved.

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Figures

Figure 1
Figure 1. Kinetics of proteolysis of wild-type bacteriorhodopsin by subtilisin

Kinetics of proteolysis of bR by subtilisin was determined under a native condition at 0.60 XSDS (●), an SDS-denaturing condition at 0.83 XSDS (○), and at 0.75 XSDS, the observed Cm by pulse proteolysis (▽). bR was pre-equilibrated in 10 mM sodium phosphate buffer (pH 6.0) containing 15 mM DMPC, 16 mM CHAPSO for at least 1 hr. bR was then diluted into the same buffer containing the designated XSDS to a final protein concentration of 0.10 mg/ml and allowed to equilibrate for 3 min. Following the equilibration, varying concentrations of subtilisin were added to each reaction. Reactions were quenched by the addition of PMSF to the final concentration of 13 mM at designated time points. The kinetic constants (kp) were determined by fitting the disappearance of intact bR over time on an SDS-PAGE gel to a first-order rate equation. The kcat/Km values at 0.60 XSDS and 0.83 XSDS were determined from the slope of the plots.

Figure 2
Figure 2. Pulse proteolysis of wild-type bacteriorhodopsin

(A) A representative SDS-PAGE gel following pulse proteolysis of bR is shown. bR was pre-equilibrated in 10 mM sodium phosphate buffer (pH 6.0) containing 15 mM DMPC, 16 mM CHAPSO for at least 1 hr. bR was then diluted into the same buffer containing varying concentrations of SDS. Reactions were incubated for 3 min before the initiation of pulse proteolysis by the addition of subtilisin to 50 μg/mL. After 1 min, reactions were quenched by the addition of PMSF to 10 mM. Quenched reactions were then analyzed by SDS PAGE. Undigested bR (−) is shown for comparison. (B) fN of wild-type bR in SDS was determined by pulse proteolysis (○) and by A560 (●). The fN values were determined from pulse proteolysis by dividing the remaining intact bR intensities by the intensity of the upper baseline (I0) value derived from the fitting of the data set to the two-state equilibrium unfolding model. Equilibrium unfolding of bR was monitored by A560 as previously described. Briefly, bR was pre-equilibrated in 10 mM sodium phosphate buffer (pH 6.0) containing15 mM DMPC, 16 mM CHAPSO for at least 1 hr. bR was then diluted into the same buffer to a final concentration of 0.10 mg/mL. The protein was titrated with 10 mM sodium phosphate buffer (pH 6.0) containing 20% SDS, 15 mM DMPC, 16 mM CHAPSO, to raise the XSDS. Following each addition of the solution, the reaction was stirred for 3 min in the dark before the A560 was read. Data was fit to a two-state equilibrium unfolding model. The XSDS at which half of the protein is denatured (Cm) determined by pulse proteolysis and A560 are 0.753 ± 0.003 XSDS and 0.732 ± 0.001 XSDS, respectively.

Figure 3
Figure 3. Effect of mutations on the denaturation of bR by SDS monitored by pulse proteolysis and A560

Denaturation of wild-type (●), M56A (▽), and Y57A (■) bR by SDS was monitored by pulse proteolysis and A560. (A) Pulse proteolysis was performed for M56A and Y57A bR as described in Figure 2. Quenched reactions were then analyzed by SDS PAGE. The intensity of remaining mutant bR after pulse proteolysis is converted to fN and plotted against XSDS for each reaction. (B) The A560 values of bR were also converted to fN and plotted against XSDS. Reactions were performed as described in Figure 2. Wild-type bR data are shown for comparison.

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