Site-Specific Information on Membrane Protein Folding by Electron

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Site-Specific Information on Membrane Protein Folding by Electron Spin Echo Envelope Modulation Spectroscopy )

Aleksei Volkov,†, Christoph Dockter,‡,^ Yevhen Polyhach,§ Harald Paulsen,‡ and Gunnar Jeschke*,§ †

Max Planck Institute for Polymer Research, Postfach 3148, 55021 Mainz, Germany, ‡Institute of General Botany, Johannes Gutenberg University Mainz, M€ ullerweg 6, 55099 Mainz, Germany, and §Laboratory for Physical Chemistry, ETH Z€ urich, Wolfgang-Pauli-Strasse 10, 8093 Z€ urich, Switzerland

ABSTRACT Compared to folding of soluble proteins, folding of membrane proteins is complicated by the fact that it requires an amphiphilic environment. Few existing techniques can provide structurally resolved information on folding kinetics. For the major plant light harvesting complex LHCII, it is demonstrated that changes in water accessibility of a particular amino acid residue can be followed during folding by measuring the hyperfine interaction of spin labels with deuterium nuclei of heavy water. The incorporation of residue 196 into the hydrophobic core of a detergent micelle was investigated. The technique provides a time constant that is similar to the one found with fluorescence spectroscopy for the slower folding step of the whole protein and with electron paramagnetic resonance for change of the distance between residues 90 and 196. If applied to several residues, this technique should provide information on the sequence of events during membrane protein folding. SECTION Biophysical Chemistry

can be characterized by a range of different techniques.9 Among these techniques, electron spin echo envelope modulation spectroscopy (ESEEM)10 in the presence of heavy water11 provided the most quantitative results that could be interpreted in the most straightforward fashion. In this ESEEM experiment, contrast between the protein, lipids, and detergents on the one side and water on the other side is introduced by deuteration of the water. Local water concentration is measured by selectively detecting the hyperfine interaction between the electron of a spin label and deuterium nuclei. Here we explore whether this experiment is suitable for measurements of membrane protein folding kinetics with structural resolution. The experiments are performed on LHCII, a complex of a 25 kDa membrane protein with eight chlorophyll a, six chlorophyll b, and four carotenoid molecules that collects about half of the photons used in oxygenic photosynthesis of green plants.12 LHCII exists in both monomeric and trimeric forms. Monomeric LHCII is transiently accumulated during the biogenesis of the photosynthetic apparatus and then assembled into trimers.13 Moreover, the dissociation of trimeric LHCII into monomers has been proposed to occur during state transition, a key event in the regulation of photosynthesis.14 Crystal structures exist only for the trimeric form,12,15 while both forms can be

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urrent knowledge on the driving forces and mechanisms of protein folding owes much to techniques that can characterize the folding pathway with structural resolution.1 The most popular of these methods monitor changes in water accessibility of amino acid residues by observing changes in the rate of amide proton/deuterium exchange by either mass spectrometry2 or liquid-state NMR.3 More diverse information on changes in residue accessibility can be obtained at improved resolution by a 19F NMR probe technique that involves modification of certain amino acids.4 However, not all classes of proteins are accessible to these and similar techniques. In particular for membrane proteins, which play central roles in energetics of living cells and in controlling substrate transport through cell membranes, folding is poorly understood.5 Difficulties arise in applying established techniques for structure determination while maintaining the complex solvent interactions that stabilize the native structure. Furthermore, the typical size of these proteins is not well suited for current NMR techniques. By combining site-directed spin labeling6 and pulse electron paramagnetic resonance techniques for distance measurements,7 slow folding processes of membrane proteins can be monitored with structural resolution, as we have recently demonstrated on major plant light harvesting complex LHCII.8 Such measurements can characterize formation and large-scale rearrangement of transmembrane helices; however, they do not provide information on changes in local solvent accessibility. On the other hand we have shown that water accessibility of residues in detergent-solubilized LHCII

r 2010 American Chemical Society

Received Date: December 17, 2009 Accepted Date: January 15, 2010 Published on Web Date: January 21, 2010

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DOI: 10.1021/jz900424n |J. Phys. Chem. Lett. 2010, 1, 663–667

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Figure 1. Derivation of the water accessibility parameter Π(D2O) from the primary ESEEM data at a folding time of 32 s. (a) Normalized experimental primary ESEEM data (black curve) and simulated data (red). An 11th-order background polynomial was fitted, subtracted from the data, and the data was then divided by this polynomial to yield the pure ESEEM modulation. (b) Pure ESEEM modulation (black) and fit by a Gaussian-damped oscillation at the deuterium frequency, D(t) = kD cos(2πνDT þ φ) exp(-T2/τ02), where kD, τ0, and φ are fit parameters. Dashed red lines indicate the total modulation amplitude. The solid and dashed red lines in the left plot (a) are obtained by adding unity and multiplying with the background polynomial.

the value Π(D2O)=0.088 for V196r obtained under folding conditions as the reference for the completely folded state. To properly interpret changes in water accessibility as a function of folding time, information is required on the position of the electron spin with respect to the protein backbone and of its spatial distribution. To obtain this information, we performed rotamer library simulations18 of the spin label conformation as well as DEER measurements. These simulations suggest that monomeric LHCII can be easily labeled by IA-PROXYL at residue 196 (partition function of 0.455 for the rotamer ensemble), while trimeric LHCII can only hardly accommodate the label (partition function 0.008), which is more bulky than the native valine side group. In agreement with this prediction, trimerization of the spin labeled mutant V196r proceeds with low yield. In LHCII trimers residue V196 is situated near the hydrophobic interface between LHCII molecules as is apparent in the two crystal structures.12,15 According to one of these structures12 a digalactosyl diacylglycerol (DGDG) lipid of a neighboring molecule is close to this site (Figure 2a). Of the two weakly allowed spin label rotamers in the trimer, one is strongly and the other one slightly oriented toward the center of the trimer (see Figure S1 in the Supporting Information). This is also apparent in the predicted V196r/V196r0 /V196r00 distance distribution, which features two peaks at distances shorter than the CR-CR distance (Figure S2 in the Supporting Information). In a four pulse double electron electron resonance (DEER) measurement19 only the distances around 1.8 nm are expected to contribute to the signal.20,21 The predicted signal, taking into account all distances longer than 1.5 nm, is in fair agreement with the measured signal (Figure S2). The presence of the short distances is revealed in the field-swept echo detected EPR spectrum that is significantly broadened for V196r LHCII trimers compared to monomers (see Figure S3 in Supporting Information). Such a spectrum should always be recorded and checked for broadening to avoid short distances with suppressed DEER modulations being missed. The trimer spectrum was simulated by convolution of the monomer spectrum with the dipolar spectrum that arises for the predicted distance distribution. The magnitude of broadening in this spectrum is in agreement with the

reconstituted into detergent micelles starting from unfolded apoprotein16 and are accessible to site-directed spin labeling.17 Under the conditions used in our folding study LHCII monomers are formed. We characterize water accessibility by the dimensionless parameter Π(D2O), introduced in ref 9, which is given by 2 2kD νD ΠðD2 OÞ ¼ ½1 -cosð2πνD τÞ 3 2 MHz where kD is the modulation depth (see Figure 1), νD the deuterium Zeeman frequency, and τ the interpulse delay between the first two pulses in the three-pulse ESEEM experiment. This definition corrects for the dependence of the modulation depth on magnetic field and interpulse delay. For 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-pyrrolidinyl1-oxyl (IA-PROXYL) spin labels in LHCII monomers this ESEEM water accessibility parameter Π(D2O) varies between 0.277 for the most water accessible site at the stromal end of transmembrane helix H1, probed by the mutant S59r and 0.065 for the least accessible site, probed by the mutant V196r.9 The mutant notation corresponds to serine 59 and valine 196, respectively, mutated to cysteine and spin labeled with IA-PROXYL. These values compare to 0.343 for the free spin label in an aqueous buffer. Reproducibility of Π(D2O) for independent preparations of the same spin labeled mutant of LHCII is about ( 0.02, except at very flexible positions, such as S160r and S3r. For different measurements of the same protein preparation reproducibility is about (0.01, with the main error source being noise. As time-resolved ESEEM experiments require a different reconstitution procedure8 than the one used in our previous water accessibility study9 and as the protein complex cannot be purified after partial folding, we also tested how well the data for purified folded LHCII can be reproduced under the experimental conditions of time-resolved ESEEM experiments with a very long folding time. Under folding experiment conditions we find slightly higher water accessibilities for mutant V196r compared to purified LHCII. The values obtained under folding conditions indicate the presence of a small fraction of unfolded proteins. In the following, we use


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DGDG molecule that solvates the labeled LHCII molecule (blue ball and stick). As a result of these restrictions, the spatial distribution of the N-O bond midpoints (violet spheres) is rather narrow, particularly along the membrane normal where the root-mean-square deviation from the mean coordinate is only 1.7 Å. This relatively narrow distribution of the spatial position of the electron spin is in agreement with the narrow distance distribution between V90r and V196r observed in ref 8 and with the primary time-domain DEER data (Figure S3 in Supporting Information). It is advantageous for interpretation of the water accessibility parameter Π(D2O). In contrast to the substantial variation of Π(D2O) between different sites in the folded state, not much variation is observed in the unfolded state. In samples prepared by mock reconstitution in the absence of pigments, Π(D2O) varies only between 0.211 for the most water accessible spin label (S52r) and 0.138 for the least accessible site (V196r). Previous work suggests that some aggregation of LHCII occurs under these conditions.8 Indeed, water accessibility is larger (Π(D2O) = 0.226) for V196r in the apoprotein solubilized in lithium dodecyl sulfate (LDS) micelles. We use the latter value as the reference for the completely unfolded state in the absence of the pigment/lipid preparation. In our experiments folding was initiated by mixing the apoprotein solubilized in LDS micelles with the pigment/lipid preparation. After the folding time t, the reaction was terminated by rapidly adding an equal volume of 80% glycerol as a cryoprotectant, loading the sample into an EPR tube and flash-freezing it in liquid nitrogen. The time resolution of this technique is limited by the addition of the cryoprotectant and the freezing time in the tube, but suffices for the folding time scale of tens of seconds to several minutes established by previous fluorescence22,23 and DEER8 experiments. Less protein-pigment mixture is consumed than with faster freeze-quench techniques. The series of ESEEM spectra obtained after different folding times is shown in Figure 3a. Intensity of the deuterium peak at a frequency of about 2 MHz decreases continuously, while intensity of the proton peak around 15 MHz remains constant. The proton peak stems from hydrogen atoms on the label, on LHCII and on alkyl chains of detergent or lipid

one in the experimental spectrum, although significant differences in the detailed lineshapes are seen (Figure S3). Such differences are expected to some extent due to neglect of the unknown exchange coupling. However, we assume that part of the difference stems from uncertainties in the rotamer library predictions. Nevertheless, the broad agreement of both DEER and field-swept echo detected EPR data with the simulations indicates that the rotamer library approach predicts the conformational distribution of the spin label quite well. In detergent-solubilized monomeric LHCII, the spin label at site V196r is surface exposed to the alkyl chains of detergent or lipid molecules, and 11 out of the 108 rotamers in the library have significant populations (Figure 2b). The label conformations (red stick representation) are somewhat restricted by side groups of neighboring residues (gray ball and stick representation), Chl 3 (green ball and stick), and possibly a

Figure 2. Position of residue 196 in LHCII trimers viewed along the membrane normal from the lumenal side (a) and monomers viewed perpendicular to the membrane normal with the stromal side on top (b). Most pigment molecules are omitted for clarity. Based on PDB structure 2BHW.12 (a) The three LHCII molecules are shown in shades between green and blue, DGDG lipid molecules are shown in a ball and stick representation, and the native side group V196 is shown in a space-filling representation. (b) Allowed IA-PROXYL spin label conformations are shown in red and the position of their N-O bond midpoints as violet spheres with the radius corresponding to rotamer population. Neighboring residues and cofactors are shown in a ball and stick representation with amino acid side groups in gray, DGDG in blue, and Chl 3 in green. The positions of the headgroup layers of the detergent micelle are indicated by yellow bars.

Figure 3. Folding kinetics of LHCII assessed by changes in water accessibility at site V196r. (a) Superposition of ESEEM spectra obtained at different folding times. The peak at about 2 MHz corresponds to the deuterium nuclei of water molecules with a possible contribution from deuterated glycerol added as a cryoprotectant. (b) Dependence of the water accessibility parameter Π(D2O), which is proportional to the ESEEM amplitude, on folding time (green filled circles, left scale) and monoexponential fits, assuming that the spectrum for complete folding corresponds to times of 1800 s (black solid line) and infinite time (black dashed line). Red filled squares (right scale) are DEER folding coefficients from ref 8, quantifying the kinetics of distance change between sites V90r and V196r during folding.


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molecules. The time dependence of Π(D2O), derived by fitting the original time-domain ESEEM data as illustrated in Figure 1, is shown in Figure 3b. Note that Π(D2O) is proportional to the intensity of the deuterium peak. Within the first 32 s, corresponding to the dead time of our freeze quench method, water accessibility at site V196r drops from Π(D2O) = 0.226 to 0.149. We assign this drop to the change of the environment of the label caused by incorporation of hydrophobic pigments and lipids into the detergent micelle. Comparison with values for other labeled sites9 reveals that the value of 0.149 corresponds to a location near the border between the headgroup layer and the hydrophobic core of the micelle, in between the situations for mutants S52r (0.191) and S123r (0.118) (see also Figure S5 in Supporting Information). The water accessibility at site V196r after 32 s is slightly larger than the one found in the partially aggregated protein obtained in a mock reconstitution without pigments (Π(D2O)=0.138). For folding times between 32 and 621 s water accessibility further drops to Π(D2O) = 0.106 (Figure 3(b)). We have assigned the completely folded state with Π(D2O) = 0.088 to a folding time of 1800 s. Fitting the time dependence of water accessibility by a monoexponential decay then yields a time constant of τ = 420 ( 50 s (solid black line). If the completely folded state is assigned to infinite time, we obtain τ = 413 ( 50 s (dotted black line). These values can be compared to the time constant of about 300 s found earlier for the change in the distance between sites V90r and V196r.8 Although the values suggest that water accessibility changes more slowly than distance, superposition of the experimental data points (red filled circles and green filled squares) reveals that the difference is not significant. On the other hand the change in water accessibility is significantly slower than the change in distance between sites S106r and S160r that has an upper limit of 50 s for the time constant.8 In our previous work we assigned the faster change to helix formation, possibly coupled with mainly chlorophyll a binding and the slower change to mutual arrangement of helices, possibly coupled with mainly chlorophyll b binding.8 It is plausible that the final position of residue 196 in the micelle is established only after the proper arrangement of helices is realized. For studying protein folding, the ESEEM technique is generally applicable if the water accessibility in the folded state differs significantly from the water accessibility after addition of pigments and lipids to the LDS solubilized apoprotein. Among the sites tested so far,9 this applies mainly to those that are poorly water accessible in the folded state, although we would also expect that an increase of water accessibility is visible for sites that are strongly exposed to water in folded LHCII. To conclude, we have shown that a combination of sitedirected spin labeling, a simple freeze-quench technique, and ESEEM spectroscopy in the presence of heavy water allows measurements of membrane protein folding kinetics, provided that the protein can be reconstituted into detergent micelles and that the folding proceeds on time scales of tens of seconds to minutes. Faster processes could be characterized by optimization of the freeze-quench technique. Work along


these lines is now in progress. By applying deuterated lipids24 the technique could also be applied to sites that are situated near the lipid headgroup layer in the folded state. Proteins used in this study were derivatives of the Lhcb1*2 (AB80) gene from pea (Pisum sativum)25 with its single cysteine in position 79 replaced with serine and valine 196 replaced with cysteine. The derivative was constructed by using the QuikChange mutagenesis kit (Stratagene). Bacterial expression of the Lhcb1 gene derivatives26 and labeling of the protein with PROXYL spin labels [3-(2-iodoacetamido)-PROXYL, Aldrich] were performed as described elsewhere.9 Reconstitution for time-resolved ESEEM experiments followed the same procedures as used for time-resolved DEER experiments in ref 8, with the only difference that all the buffers were prepared with deuterated water and glycerol-d8 was used for cryoprotection. Trimer samples were prepared as described in ref 17. The X-band (9 GHz) pulse EPR measurements were performed on a Bruker Elexsys EX 580 EPR spectrometer using a Bruker Flexline split-ring resonator ER 4118X_MS3 overcoupled to Q ∼ 100. All pulse measurements were performed at 50 K with liquid helium cooling using an Oxford CF935 cryostat with an Oxford ITC4 temperature controller. The four-pulse DEER experiment19 was performed with an interpulse delay τ2 of 800 ns, a repetition time of 6 ms and a total measurement time of 12 h. Field-swept electron spin echo (ESE) and ESEEM measurements were performed and analyzed as described in ref 9. The Matlab program for analysis is available from the authors on request. Rotamer simulations at a target temperature of 175 K, roughly corresponding to the glass transition temperature of the sample, and spin label visualization were done with the Matlab (The MathWorks, Natick, MA) program package MMM which is freely available at http://www.epr.ethz.ch/software/ index.

SUPPORTING INFORMATION AVAILABLE Method for simulation a field-swept echo-detected EPR spectrum for a trimer sample from the distance distribution. Figures: (S1) Fieldswept echo-detected EPR spectra of V196r monomers and trimers. (S2) Experimental test of the rotamer prediction for the conformational distribution of the IA-PROXYL spin label attached to residue V196C in LHCII trimers. (S3) Comparison of experimental and simulated primary DEER data for LHCII monomers labeled at residues 90 and 196. (S4) Predicted rotamers for site V196r in LHCII trimers. (S5) Predicted rotamers for sites S52r, S123r, and V196r in LHCII monomers. This information is available free of charge via the Internet at http:// pubs.acs.org.

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. Phone: þ41 44 632 5702. Fax: þ41 44 633 1448. E-mail: gunnar.jeschke@phys. chem.ethz.ch.

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Present Addresses: Current address: BASF SE, 67063 Ludwigshafen, Germany. Current address: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.

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ACKNOWLEDGMENT We thank H. W. Spiess for providing measurement time on the pulse EPR spectrometer, C. Bauer for technical support, and M. Hebel for his help with constructing some of the LHCII derivatives. Deutsche Forschungsgemeinschaft (SFB 625, TP B 10) is acknowledged for funding.

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Site-Specific Information on Membrane Protein Folding by Electron

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