Insights into Cotranslational Membrane Protein Insertion by Combined

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Insights into co-translational membrane protein insertion by combined LILBID-mass spectrometry and NMR spectroscopy Oliver Peetz, Erik Henrich, Aisha Laguerre, Frank L#hr, Christopher Hein, Volker Dötsch, Frank Bernhard, and Nina Morgner Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03309 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Analytical Chemistry

Figure 1. Modulation of PR complex formation. PR was cell-free expressedsynthesized in the presence of increasing MSP1E3D1 (DMPG) nanodisc concentrations (from bottom to top: 100 µM, 60 µM, 20 µM, 10 µM). The complexes were purified by C-terminal StrepII-tag of PR and analyzed by LILBID-MS. Complexes corresponding to the detected signals are indicated by pictograms. Spectra were recorded by low (10 mJ) laser power (a) and high (23 mJ) laser power (c). (b) and (d) show the normalized quantification of the corresponding peak areas. The grey bars in (b) indicate the initially adjusted PR:nanodisc ratio, while the light purple bars represent the average final PR:nanodisc ratio (i.e. oligomerisation state) after purification as determined by LILBID. See supporting information for a zoom-in of the higher mass complexes of (c) . (For reference: Sspectra of 6,6:1 ratio see 8 611x433mm (300 x 300 DPI)

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Figure 2. Nanodisc titration of KcsA and LspA expression. KcsA was cell-free expressed synthesized in the presence of 20 µM and 50 µM MSP1E3D1 (DMPG) nanodiscs, resulting into KcsA:nanodisc ratios of approximately 2.5:1 and 1:1. Samples were purified by the C-terminal StrepII-tag of KcsA and analyzed. (a) LILBID-MS analysis of KcsA nanodiscs at low (10 mJ) laser power. Complexes corresponding to the detected signals are indicated by pictograms. (b) SDS-PAGE analysis and Coomassie-Blue staining of the purified KcsA nanodiscs. LspA was cell-free expressed synthesized in the presence of 20 µM, 50 µM and 100 µM MSP1E3D1 (DMPC) nanodiscs (ND), resulting into LspA:nanodisc ratios of approximately 2.5:1 to 0.5:1. Samples were purified by the C-terminal StrepII-tag of LspA and analyzed (c) by LILBID-MS at high (23 mJ) laser power. Spectra at 17 or 10 mJ also revealed only monomeric LspA and lower laser power resulted into low resolution (data not shown) (See supporting information Fig 2). Complexes corresponding to the detected signals are indicated by pictograms. No indication of insertion of a second LspA into a nanodic was found. 408x416mm (300 x 300 DPI)


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Figure 3. Illustration of the proposed cooperative membrane insertion mechanism of KcsA involving polysomes. The proximity of the ribosomes allows for interaction of an already inserted monomer and the next nascent chain (inset), guiding the folding and insertion process into the same nanodisc. 408x189mm (300 x 300 DPI)



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Oliver Peetz1‡, Erik Henrich2‡, Aisha Laguerre2, Frank Löhr2, Christopher Hein2, Volker Dötsch2, Frank Bernhard2, Nina Morgner1* 1

Institute of Physical and Theoretical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany

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Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt am Main, Germany *Correspondence goes to: [email protected] ABSTRACT: Co-translational insertion of membrane proteins into defined nanoparticle membranes has been developed as an efficient process to produce highly soluble samples in native-like environments and to study lipid dependent effects on protein structure and function. Numerous examples of structural and functional characterization of transporters, ion channels or G-protein coupled receptors in co-translationally formed nanodisc complexes demonstrate the versatility of this approach, although the basic underlying mechanisms of membrane insertion are mainly unknown. We have revealed first aspects of the insertion of proteins into nanodiscs by combining cell-free expression, non-covalent mass spectrometry and NMR spectroscopy. We provide evidence of cooperative insertion of homooligomeric complexes and demonstrate the possibility to modulate their stoichiometry by modifying reaction conditions. Additionally, we show that significant amounts of lipid are released from the nanodiscs upon insertion of larger protein complexes.

The recently emerging combination of cell-free expression with the nanodisc technology provides excellent synergies for the detergent-free production of membrane proteins1-4. Preformed nanodiscs assembled with tailored lipid mixtures are supplied into cell-free reactions, and expressed membrane proteins can already cotranslationally insert into the artificial membranes5, 6. Screening and selection of appropriate lipids or lipid mixtures for the nanodisc membrane assembly can be crucial for the efficient insertion and folding of individual membrane proteins. This strategy avoids any detergent contact and allows to study even membrane proteins that could so far not be obtained in functional condition if solubilized in various detergent micelles7. However, insertion of proteins into native cellular membranes and their subsequent conformational folding into higher order assemblies often depends not only on the presence of adequate lipids, but on specific catalytic machineries such as translocon complexes. In contrast, the artificial nanodisc membranes are devoid of any translocons and the underlying insertion mechanisms of this new synthetic strategy are still completely unknown. We have recently shown that Laser Induced Liquid Bead Ion Desorption mass spectrometry (LILBID-MS) can be a valuable tool to analyse membrane protein/nanodisc complexes8. In this work, we approach the investigation of the co-translational protein insertion process into nanodisc membranes during cell-free expression. We have analyzed insertion and assembly of three different membrane proteins forming monomers, tetramers and hexamers by manipulating membrane protein to nanodisc

stoichiometry followed by complex analysis and by quantifying the resulting protein to lipid ratios. Results and Discussion Three membrane proteins appearing in different native oligomeric states were selected: Cell-free expression and the formation of the corresponding complexes in nanodisc membranes of the monomeric signal peptidase LspA9, the tetrameric potassium channel KcsA10, and the pentameric or hexameric proton pump proteorhodopsin (PR)11, a green-absorbing PR from a marine gammaproteobacterium12, was recently demonstrated8. Scenarios for the co-translational protein integration into nanodiscs could be: (i) Independent and statistical insertion of protein monomers with equal likelihood from both nanodisc sides. (ii) Individual monomers of a complex might show some cooperative insertion. Thereby, the first inserting monomer starts to fold and presents an interaction point for the nascent chain of a second monomer from a proximal ribosome in a polysome array13. This would facilitate insertion and folding of several monomers in the same nanodisc from the same side. Evidence of coordinated folding of bacteriorhodopsin complexes upon insertion into nanodiscs was recently obtained by infrared spectroscopy and a two-step folding procedure is proposed14. (iii) Complexes might already preform to some extend during translation and integrate as complete assemblies. We first analyzed whether oligomer assembly could be manipulated by modifying the concentration of provided

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Figure 1. Modulation of PR complex formation. PR was cell-free expressed in the presence of increasing MSP1E3D1 (DMPG) nanodisc concentrations (from bottom to top: 100 µM, 60 µM, 20 µM, 10 µM). The complexes were purified by C-terminal StrepIItag of PR and analyzed by LILBID-MS. Complexes corresponding to the detected signals are indicated by pictograms. Spectra were recorded by low (10 mJ) laser power (a) and high (23 mJ) laser power (c). (b) and (d) show the normalized quantification of the corresponding peak areas. The grey bars in (b) indicate the initially adjusted PR:nanodisc ratio, while the light purple bars represent the average final PR:nanodisc ratio (i.e. oligomerisation state) after purification as determined by LILBID. See supporting information for a zoom-in of the higher mass complexes of (c) (For reference spectra of 6,6:1 ratio in (a) and (c) see 8).

nanodiscs, which are preformed from two membrane scaffold proteins (MSPs) and lipids (DMPG or DMPC). See supporting information for more details. Estimated final concentration of the proteins in our expression reactions were 66-80 µM (PR), 50-55 µM (LspA) and 45-50 µM (KcsA). The concentration of KcsA and LspA were determined by immunoblot quantification and PR concentrations could be measured by the specific absorption of its retinal chromophore to provide a protein to nanodisc ratio in the reaction mixture8, 15. Nanodisc concentrations at the beginning of the expression were adjusted from 10-100 µM and protein to nanodisc ratios were thus modulated within the range from 2.5-7:1 to approximately 1:2. During the purification process of the corresponding samples the empty nanodiscs are removed. Therefore a change in average oligomeric complex size in a filled nanodisc will lead to a different molar ratio of protein to nanodiscs. (Fig. 1). LILBID-MS spectra recorded

under mild conditions show a high fraction of PR tetra-, penta- and hexamers with 10 and 20 µM (6.6:1 and 3.6:1) nanodiscs, but mainly PR dimers and trimers at 100 µM (0.83:1) nanodiscs (Fig. 1a). This becomes even more evident when considering the peak integrals of the mass spectra depicted in Figure 1b. Comparison of spectra and peak intensities at different ratios both at low (Fig. 1a, b) and high laser power (Fig. 1c, d) reveals the general trend of a reduced PR assembly complexity with increasing nanodisc concentrations. High laser power results in increased disintegration of the PR nanodisc particles, revealing among others isolated PR complexes up to hexamers, indicating their stability. Interestingly, no higher oligomeric state than the hexamer is detected even at PR to nanodisc ratios of 6.6:1 (Fig. 1). The results clearly exclude the possibility of preformed complexes entering the nanodisc, as we see different oligomeric distributions depending on the PR:nanodisc ratio. However, insertion

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stoichiometry was lower as initially adjusted in the reaction.

Figure 2. Nanodisc titration of KcsA and LspA expression. KcsA was cell-free expressed in the presence of 20 µM and 50 µM MSP1E3D1 (DMPG) nanodiscs, resulting in KcsA:nanodisc ratios of approximately 2.5:1 and 1:1. Samples were purified by the C-terminal StrepII-tag of KcsA and analyzed. (a) LILBIDMS analysis of KcsA nanodiscs at low (10 mJ) laser power. Complexes corresponding to the detected signals are indicated by pictograms. (b) SDS-PAGE analysis and Coomassie-Blue staining of the purified KcsA nanodiscs. LspA was cell-free expressed in the presence of 20 µM, 50 µM and 100 µM MSP1E3D1 (DMPC) nanodiscs (ND), resulting into LspA:nanodisc ratios of approximately 2.5:1 to 0.5:1. Samples were purified by the C-terminal StrepII-tag of LspA and analyzed (c) by LILBID-MS at high (23 mJ) laser power. Spectra at 17 or 10 mJ also revealed only monomeric LspA (See supporting information Fig 2). Complexes corresponding to the detected signals are indicated by pictograms. No indication of insertion of a second LspA into a nanodic was found.

appears not to be purely statistical but rather indicates a certain cooperativity as PR oligomers already dominate at initially low PR:nanodisc ratios. Comparison of the finally measured molar stoichiometry of the purified PR:nanodisc assemblies with the initially adjusted ratios in the crude reaction revealed final ratios of 2.0:1 (initial ratio was 0.83:1), 2.2:1 (initial 1.2:1), 3.8:1 (initial 3.6:1) and 4.3:1 (initial 6.6:1) as determined from the low laser power LILBID-MS spectra by peak integration (Fig. 1b). This indicates that at excess of supplied nanodiscs already in average a PR dimer is integrating while many nanodiscs remain empty and are removed during purification. A PR:nanodisc ratio of approx.. 3.6:1 appears to generate an optimal balance in between membrane insertion and translation efficiency. Further limitation of nanodiscs resulted into increased precipitation of produced PR, most likely due to the failure of numerous nascent polypetides to contact a nanodisc membrane. Accordingly, the finally determined average PR

Further support of cooperative insertion was obtained by the analysis of KcsA expressed with different nanodiscs concentrations (Fig. 2). With 20 µM nanodiscs, giving roughly a calculated molar ratio of KcsA:nanodiscs of 2.5:1, still exclusively KcsA tetramers were detected in the purified sample at mild laser conditions (Fig.2a). Increased nanodisc concentrations resulted in an oligomeric distribution for KcsA from monomer to tetramer, with the tetramer providing now only 22,0% of the KcsA species. The exclusive formation of a KcsA tetramer in Fig.2a is a clear indication that the protein is properly folded, allowing for the assembly into the KcsA specific quaternary structure. The lower oligomers at higher nanodisc concentrations represent KcsA proteins en route to the formation of tetramers. Noteworthy here is the unusual strength of the interactions between the KcsA proteins in a tetramer, which was further verified by SDS-PAGE analysis, showing tetramers while dimers and trimers do not stay stable during that treatment (Fig. 2b). In contrast, the monomeric signal peptidase LspA stayed monomeric in all LspA:nanodisc ratios from 1:2 (100 µM nanodiscs) until 2.5:1 (20 µM nanodiscs) (Fig. 2c). Multimeric LspA complexes could not be detected with LILBID-MS. Even though it should be possible that multiple LspA monomers integrate into one nanodisc, in particular at high LspA:nanodisc ratios no complexes with more than one LspA are detected. The combined data indicate that cooperative mechanisms are involved in the membrane integration process of PR and KcsA, most likely enabled by polysome arrays formed in the cell-free reaction13, 16 (Fig. 3). This will place evolving nascent monomers into close proximity of already inserted and folded or still folding monomers. Initiation of specific interface formation in between the monomers would then support their multiple insertion into the same nanodisc by (i) interaction-guided insertion as well as by (ii) prolonged tethering of the nanodisc to the polysome. Strength and time point of possible interactions during the monomer folding process are certainly protein

Figure 3. Illustration of the proposed cooperative membrane insertion mechanism of KcsA involving polysomes. The proximity of the ribosomes allows for interaction of an already inserted monomer and the next nascent chain (inset), guiding the folding and insertion process into the same nanodisc.

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Analytical Chemistry loaded MSP1D1ΔH5 (DMPC) nanodiscs resulting from cellfree expression reactions with PR to nanodisc ratios of 1.8:1 and 2.7:1 determined in the purified samples (Fig. 4a). Lipid signals are detectable by standard phosphorous NMR based on the presence of one 31P atom within the DMPC headgroups. The lipid signals were normalized to the total nanodisc concentration via 13C, 1D experiments by using 15N, 13C labeled MSP1D1ΔH5 for nanodisc formation. Comparison of the normalized lipid concentrations before and after PR insertion reveals a gradual reduction of the lipid signal of approximately 30% at the ratio of 2.7:1 (Fig. 4b and c). This correlates nicely with the calculated space of 16.2 nm2 required from the average of 2.7 PR at the analyzed ratio, suggesting the release of a corresponding number of lipids (Fig. 4d).

Figure 4. Lipid release of nanodisc membranes upon PR insertion. PR was expressed in the presence of MSP1D1ΔH5 (DMPC) nanodiscs pre-formed with 15N, 13C labeled MSP1D1ΔH5. The samples were purified by the C-terminal StrepII-tag of PR and PR:nanodisc ratios were determined by LILBID-MS at 1.8:1 and 2.7:1. Samples were analyzed by phosphorous and by 13C 1D NMR. (a) LILBID-MS spectra of the two samples at 15 mJ. (b) Overlay of DMPC signals obtained by phosphorous NMR. (c) Normalized quantification of DMPC content in the samples. (d) Scheme of PR insertion into nanodisc membranes concomitant with lipid release.

dependent and will yield protein specific complex stoichiometry such as preferred tetramers for KcsA and multiple oligomeric forms for PR. Higher nanodisc concentrations will then gradually increase the competition in between independent membrane insertion and interaction-guided insertion, resulting in the observed reduced statistical complex stoichiometry. Nanodisc insertion is principally possible from both sides, but the predominant formation of stable KcsA tetramers indicates the presence of all monomers in one orientation. Besides interaction-guided insertion, this uniform orientation might be further supported by rapid flipping of protein monomers within the membrane17 until the favorable stable oligomeric complexes have been formed. Inhomogeneities at the interface of membrane scaffold proteins and lipid bilayer might favor such flipping events. The remaining question was the impact on the nanodisc membrane itself upon insertion of e.g. a large PR complex. One PR pentamer (5x6 nm2) occupies approximately 5055% of the available membrane space of a MSP1D1ΔH5 nanodisc (~55 nm2)11, 18. Re-organization of the membrane lipids around the inserted protein, release of a certain number of lipids or expansion of the whole nanodisc19 due to a dynamic structure of the scaffold proteins20 are discussed as possible scenarios. To address this question, we performed 1D-NMR experiments with empty and PR

We present here an experimental approach that allows investigating the process of translocon free protein insertion into a membrane. While the number of subunits in higher oligomeric complexes can be reduced by modifying the nanodisc stoichiometry, the induction of artificial multimerization appears not to be possible with the analyzed proteins. For insertion of homooligomeric complex forming proteins into artificial nanodisc membranes, our results underpin the model of cooperative support presumably provided by polysome formation and/or complex specific monomer interactions already during the protein insertion process (Fig. 3). The results agree with current models of membrane protein insertion. Thermodynamically the protein insertion into membranes with and without the translocon machinery is the same21. The translocons’ main function may rather be to coordinate insertion into the densely packed cell membrane22. Translocon-free insertion is thus possible in the much less complex nanodisc environment and potentially supported by their mobility, topology and two-sided access. Insertion of proteins into nanodisc membranes finally causes release of a corresponding number of lipids presumably in form of small vesicles, possibly in concert with a previous limited expansion of the nanodisc particle. More work will be required to separate protein-protein interaction from the restraining effects of the nanodisc, especially where protein integration might be hampered by size limits of the nanodiscs. This might in future allow for quantitative analysis, revealing KD values and allowing optimization of cell-free protein nanodisc complex formation with regard to scaffold protein, lipid mixture or cell-free expression time.

* Nina Morgner ([email protected])

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The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.

E.H., C.H. and O.P. are supported by the DFG (German Research Foundation), Collaborative Research Center 807 „Transport and Communication across Biological Membranes“ A.L. is funded by the German Research Foundation (DO545/11) V.D. and N.M. are supported by Cluster of Excellence Frankfurt (Macromolecular Complexes) N.M. received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC Grant agreement n° 337567.

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Insights into Cotranslational Membrane Protein Insertion by Combined

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