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Cell-Free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility

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Cell-Free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure Article Cell-free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility Manuel Etzkorn,1 Thomas Raschle,1 Franz Hagn,1 Vladimir Gelev,3 Amanda J. Rice,2 Thomas Walz,2 and Gerhard Wagner1,* 1Department of Biological Chemistry and Molecular Pharmacology 2Department of Cell Biology Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA 3FB Reagents, 267 Pearl Street, Cambridge, MA 02139, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.01.005 SUMMARY polymers (amphipols) (Popot et al., 2011) or lipid bilayer nano- discs (Denisov et al., 2004) have lately received increased atten- Selecting a suitable membrane-mimicking environ- tion as promising tools for the investigation of membrane ment is of fundamental importance for the investiga- proteins (Gorzelle et al., 2002; Raschle et al., 2010; Zoonens tion of membrane proteins. Nonconventional surfac- et al., 2005). Advantages of using nonconventional membrane tants, such as amphipathic polymers (amphipols) mimetics include the exceptionally good refolding properties and lipid bilayer nanodiscs, have been introduced of amphipols for heptahelical membrane proteins such as as promising environments that may overcome G-protein-coupled receptors (GPCRs) (Dahmane et al., 2009) or the absence of detergent as well as the more native-like envi- intrinsic disadvantages of detergent micelle sys- ronment provided by nanodiscs. Additionally it has been demon- tems. However, structural insights into the effects strated that the use of nonconventional surfactants can increase of different environments on the embedded protein protein stability and improve the accessibility of the functional are limited. Here, we present a comparative study form (Popot, 2010). Initial NMR studies on b-barrel proteins indi- of the heptahelical membrane protein bacteriorho- cate that amphipols (Zoonens et al., 2005) as well as nanodiscs dopsin in detergent micelles, amphipols, and nano- (Glu¨ ck et al., 2009; Raschle et al., 2009; Shenkarev et al., 2009) discs. Our results confirm that nonconventional may also be useful for NMR structural studies of polytopic environments can increase stability of functional membrane proteins. However, recent results obtained for a class bacteriorhodopsin, and demonstrate that well- A GPCR suggest that high-resolution solution-state NMR struc- folded heptahelical membrane proteins are, in prin- tural studies of heptahelical membrane proteins in nanodiscs are ciple, accessible by solution-NMR methods in restricted to the extramembranous part of the protein (Park et al., 2011). Moreover, our fundamental understanding of the effects amphipols and phospholipid nanodiscs. Our data that different membrane mimetics have on protein structure distinguish regions of bacteriorhodopsin that medi- and dynamics is still very limited. ate membrane/solvent contacts in the tested envi- The heptahelical transmembrane protein bacteriorhodopsin ronments, whereas the protein’s functional inner (bR) offers ideal biophysical properties such as molecular size, core remains almost unperturbed. The presented topology, stability as well as its characteristic color (indicative data allow comparing the investigated membrane of intact tertiary structure) to study the effects of different mimetics in terms of NMR spectral quality and membrane mimetics on its structure and stability. As a light-acti- thermal stability required for structural studies. vated proton pump bR consists of two moieties, the 27 kDa protein bacterioOpsin (bO) and the retinal, a vitamin A metabo- lite, covalently bound to a lysine side chain of bO. Due to its INTRODUCTION exceptionally high abundance as part of the purple membrane in Halobacterium salinarum, structural studies of bR so far The in vitro investigation of membrane protein structure and have been solely carried out on the functional protein extracted function is inherently related to the choice of a suitable from its native environment. The bR structure itself has been membrane-mimicking environment. While great progress has extensively studied, in particular, by X-ray and electron crystal- been made in the elucidation of membrane protein structure in lography (Edman et al., 1999; Hirai and Subramaniam, 2009; detergent micelles using solution-state nuclear magnetic reso- Luecke et al., 1998, 1999a, 1999b; Oka et al., 2000), as well as nance (NMR) (Kang and Li, 2011; Kim et al., 2009; Nietlispach solid-state NMR (Bajaj et al., 2009; Engelhard et al., 1989; and Gautier, 2011), detergents are often detrimental to protein Harbison et al., 1983, 1984; Saitoˆ et al., 2000). Additionally, structure and may not (fully) support its functional form, in partic- a partial resonance assignment of bR in n-dodecyl-b-D-malto- ular, if soluble domains are present that may be unfolded by pyranoside (DDM) micelles using solution-state NMR tech- detergents. Nonconventional surfactants such as amphipathic niques has been published (Schubert et al., 2002). While 394 Structure 21, 394–401, March 5, 2013 ª2013 Elsevier Ltd All rights reserved Structure NMR on bR in Nonconventional Membrane Mimetics RESULTS AND DISCUSSION Effects of Cofactor-free In Vitro Expression Initially, we compared the resonance assignments of bR in DDM micelles extracted from the native membrane (Schubert et al., 2002) to the cell-free expressed and refolded bR (also in DDM micelles) (see Figure S1available online). The close similarity of the observed peak positions strongly indicates that our re- folded protein closely resembles the tertiary structure of the membrane-extracted protein and allows a direct transfer of most peak assignments (Schubert et al., 2002). Interestingly, a cluster of noticeable chemical-shift alternations is found for residues around Lys 172 (Figures 1 and S1). In the crystal struc- ture (Luecke et al., 1999b), direct interactions of the Lys 172 and Val 173 side chains to the head groups of (cocrystallized) lipids are found. Since these lipids are absent in the in vitro expression system, our data suggest that specific lipids are copurified when bR is extracted with detergents from the purple membrane (e.g., lipid 1 and/or lipid 2 facing helix F-G but most likely not lipid 3 Figure 1. The Role of Cell-free Expression for a Bias-free Analysis of facing helix A-G; Figure 1). This demonstrates the importance the Environment of using a well-defined system, such as offered by in vitro This figure shows differences between bR in DDM micelles when extracted expression, to enable a bias-free investigation of the effects of from the native purple membrane (Schubert et al., 2002) or when refolded after different membrane mimetics on bR structure and stability. cell-free expression. Residues in the transmembrane region that experience chemical shift alterations due to the different sample preparation are high- lighted in red (on the crystal structure; Luecke et al., 1999b). Blue residues do Biophysical Properties of the Different Membrane not display any significant difference. Lipids (fragments) as present in the crystal Mimetics are shown in yellow. See Figure S1 for experimental data and more details. Figure 2 summarizes biophysical properties of cell-free ex- pressed bR in the tested environments. Size-exclusion chroma- extraction of/from the native purple membrane is a very powerful tography (SEC) clearly indicates that the particle size of bR in strategy, it has the downside that cofactors such as the cova- detergent micelles (bR-DDM) is significantly smaller (about lently bound ligand or coordinated lipids are often copurified 40%) than in the amphipol (bR-APOL) and nanodiscs (bR-ND) (Patzelt et al., 1997). The latter will interfere with the measure- environments, which themselves are comparable in size (Fig- ments of effects of different membrane-mimicking environments ure 2A). However, bR-ND has a more homogenous size dis- on protein structure and function by introducing a bias most tribution than bR-APOL. Interestingly, thermal denaturation, likely toward the native conformation. Heterologous cell-free measured by thermofluor assays (Matulis et al., 2005), shows protein expression offers an interesting option for the production that the micelle offers the least stable environment for bR (Fig- of ‘‘cofactor-free’’ bO (Cappuccio et al., 2008). ure 2B). We also measured the lifetimes of functional bR by We present a comparative study of bR in DDM micelles, A8-35 time-resolved absorption spectroscopy at 58C(Figure 2C). It amphipols (Popot et al., 2011) as well as in lipid bilayer nanodiscs is clearly evident that the functional state of bR is best preserved formed using 1,2-dimyristoyl-sn-glycero-3-phosphocholine in the lipid bilayer nanodisc environment (t1/2@58C > 14 hr), fol- (DMPC) lipids and the membrane scaffold protein MSP1D1 lowed by the amphipols (t1/2@58C = 32 min) and the detergent (Denisov et al., 2004). To avoid artifacts originating from impre- micelles (t1/2@58C = 8 min). This finding is supported by the cise starting conditions (i.e., coordinated lipids), we used cell- observation that long-time storage (several months) at 5C led free methods (Cappuccio et al., 2008; Schwarz et al., 2007)to to significant reduction of absorption at 550 nm for bR-DDM express bO in the absence of detergents, lipids, or ligand. samples, while bR-APOL and bR-ND samples did not
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