Measuring membrane protein stability under native conditions

Abstract

The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.membrane protein folding | steric trap M ethods to measure the thermodynamic stability of membrane proteins have largely followed methods developed for soluble protein folding (1). The fraction unfolded is first measured as a function of denaturant concentration (urea, guanidine-HCl, etc.), which in turn provides the unfolding free energy (ΔG U ) as a function of denaturant. The fraction unfolded, however, can be accurately measured only at high denaturant concentration, where the amount of unfolded protein is large enough-the so-called transition zone. Thus, obtaining a measure of the unfolding free energy in the absence of denaturant requires extrapolation from the transition zone. For chemical denaturation, the unfolding free energy is typically linearly dependent on the denaturant concentration in the transition zone, allowing a linear extrapolation back to zero denaturant. However, although there is now considerable experimental and theoretical validation of this approach for soluble proteins (2-4), the validity of these extrapolations is not clear for measuring stability of membrane proteins.Since the observation of Braiman et al. that bacteriorhodopsin (bR) can be refolded from an SDS-denatured state (5), SDS has been commonly used to study the folding of helical membrane proteins. The Booth laboratory has pioneered and extensively studied the refolding kinetics of bR from an SDS-denatured state (6-8). We introduced SDS unfolding to measure the thermodynamic stability of the membrane enzyme diacylglycerol kinase (9) and a similar approach can be used to measure bR thermodynamic stability (10, 11). bR contains a covalently bound retinal chromophore that complicates unfolding analysis because it can slowly hydrolyze off in the SDS unfolded protein (12). We and others originally ...

Mapping the energy landscape for second-stage folding of a single membrane protein

“…Bulk SDS unfolding experiments report Δ G values from 7.08 k B T to 13.88 k B T (refs. 24,25), which is in reasonable agreement considering the completely different methods for driving unfolding, the different environments and the uncertain extrapolations in SDS unfolding studies 5 (Supplementary Table 3). Our measured refolding rate of 3.91 × 10 −2 s −1 is very similar to the rate measured in the detergent refolding experiments (2.7 × 10 −2 s −1 ), a parameter that does not require much extrapolation.…”

“…Ideally, studies of the second stage of folding would be performed in a membrane environment, yet folding studies require a means for altering the energy landscape to favor the unfolded state, which is hard to achieve in a membrane. One method, called steric trapping, drives unfolding by using a protein that binds preferentially to the unfolded state 4,5 . Atomic force microscopy (AFM) has been extensively used to study forced unfolding of membrane proteins from bilayers 6–9 .…”

Mechanisms of Integral Membrane Protein Insertion and Folding

“…The free energy of unfolding can be determined by measuring of the amount of doubly bound protein during unfolding. In this way, Chang et al determined a value of approximately −11 kcal mol −1 for the unfolding of bR [58], which is within range of values observed for water-soluble proteins. This is not a marginal value of stability.…”

Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins

“…For such efforts to be extended to other classes of membrane proteins, new experimental and theoretical frameworks will be necessary. A recent development of steric trapping method that allows the reversible control of the membrane protein folding will open new opportunities for studying the forces and mechanisms of folding of membrane proteins in their native bilayer background (Blois et al 2009 ;Chang and Bowie 2014 ;Hong et al 2010 ;Hong and Bowie 2011 ;Jefferson et al 2013 ). Hansen et al 2011 ) …”