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Quantification of Membrane Protein Self-association with a High-throughput Compatible Fluorescence Assay Junbei Li, and Xiaoyan Jade Qiu Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00009 • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on April 1, 2017
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Quantification of Membrane Protein Self-association with a Highthroughput Compatible Fluorescence Assay Junbei Li§, Xiaoyan J. Qiu*,§ §
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China KEYWORDS: membrane protein association, homo-FRET, high-throughput assay
ABSTRACT: For many membrane proteins, self-association serves both structural and functional roles. Studies of such association can be simplified by switching to micelles as the membranemimicking environment. But native interaction is not preserved in all detergents. The selection of suitable conditions for biochemical experiments would be greatly facilitated by a quantitative highthroughput assay. Here we showed that the fluorescence polarization (FP) reduction, resulted from homo-FRET and measured in a high-throughput compatible format, can be used to determine both association states and constants for membrane proteins in micelles.
Association of membrane proteins plays important roles in complex assembly and activity regulation1. But quantification of such association in living cells has been a difficult task. A relative simple approach is to study the self-association of membrane proteins with homo-FRET, which requires only one type of fluorescent label. This approach still requires either specialized instrumentation (for time-resolved anisotropy decay) or complicated experimental procedures (photobleaching2 or controlled degree of labeling3). In addition, mathematical treatment of steady-state anisotropy signals cannot distinguish between association and bystander effect without measuring the actual protein concentration4. The bystander FRET becomes quite significant at high expression level5, and cellular expression levels are usually difficult to control. The homo-FRET approach may be further simplified by switching to the membrane mimicking micelles. Detergents have been extensively used to extract, stabilize, and crystalize membrane proteins6. In micelles, protein concentrations can be well controlled, and the equilibration time is significantly reduced. Not all detergents can stabilize the native structures of membrane proteins, and this stabilization may be affected by small changes in experimental conditions (salt, pH, etc.). The best condition, that both stabilizes protein structure and retains biological functions, is currently selected via trial and error. So a high-throughput assay is instrumental in the quick screening of a large number of conditions. Several methods have been developed to measure membrane protein stability in detergents7-10. Except for the qualitative and time-consuming FSEC7, none of the methods provide information on association states. Some also places prerequisites on the protein of interests, e.g., the presence of unpaired interior Cys residue9. Therefore, we aimed to develop a FP assay that measures association quickly and quantitatively, and utilizes gen-
eral labeling procedures. In micelles, associating membrane proteins often exist in a monomer-oligomer equilibrium. The degree of association depends on protein concentration and the identity of the specific detergent. When oligomers are formed, the distance between the fluorescent labels becomes close enough for homoFRET to occur, reducing the fluorescence polarization (FP) of the label. This allows us to quantify the self-association via monitoring protein-concentration-dependent FP changes. For proof of concept, two self-associating α-helical peptides were chosen: the transmembrane regions of glycophorin A (GpA) and influenza A proton channel M2 (M2TM). GpA is dimeric with the canonical GXXXG interacting motif11. Dimerization is significantly reduced with a G to L mutation (GpAmut)12. M2TM forms a tetrameric channel, and located in the pore is the bind site for the anti-flu drug amantadine (amt)13. The drug-resistant mutant (S31N) still forms tetramer but drug binding is abolished14. In micelles, all peptides undergo monomer-oligomer equilibrium. The oligomer formation should be promoted by peptide concentration increase or drug binding to the oligomer. Any fluorophore with significant overlap between the excitation and emission spectra should have the homo-FRET property. Here nitrobenzoxadiazole (NBD) was chosen for labeling at the N-terminal of all peptides. Steady-state FP was measured in 96-well plate format with good signal-to-noise ratio, paving the way for future highthroughput assays. As membrane proteins are dissolved exclusively in micelles regardless of the buffer volume, the protein concentration here refers to the effective concentration, i.e., the absolute protein concentration divided by the absolute detergent concentration. FP value for non-interacting fluorophore is insensitive to its concentration. Therefore, monomers at the same effective concentration should have the same FP value, regardless of the absolute concentrations. This turned out to be true in the concentration range used (Table S2). The tiny deviations observed, on the order of the systematic error and negligible, most likely arose from association of labeled M2TM in solutions with low concentration of detergent. Thus we were able to fix the absolute detergent concentration well above their critical micelle concentrations (CMCs, Table S1) and gradually lower the absolute protein concentration with serial dilution. This minimizes reagents usage (less than 7.5 nmole protein for each serial dilution in 2.5 mM detergent), as well as ensures a reasonable dynamic range in effective concentrations (over 1000-fold in 2.5 mM detergent). Moreover, oligomer formation is accompanied by releasing or up-taking of detergent molecules. This changes the monomer-micelle equilibrium of the detergent solution, and in turn influences protein association equi-
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Biochemistry
Biochemistry
librium in a protein concentration dependent manner. This influence can be negligible when protein concentration is low and detergent concentration is well above CMC. For GpA and GpAmut, acidic pH was used to facilitate dissociation due to charge repulsion of the flanking Lys residues. As expected, FP value was constant for the monomeric GpAmut, while GpA displayed a shifting FP curve consistent with a monomer-oligomer equilibrium (Figure 1A). For the monomers of GpA and GpAmut, assuming micelle binding was not significantly affected by the conservative mutation, then both should have the same FP value in any detergent. Therefore, the FP values of GpA can be normalized with that of GpAmut obtained in the same detergent (Table S3). This allowed further quantification without having to obtain the FP value for the GpA monomer experimentally. The curves can be fitted well with a monomer-dimer model (Figure 1B, Table S4), implying that the NBD label did not alter the association state of GpA. Association of GpA was stronger in DM than in DDM, consistent with the literature15. We obtained Kd values of 1.25×10-5 and 1.81×10-4 in DM and DDM at pH 6, respectively. Association is tighter in neutral solutions, and Kd values of 2.16×10-6 and 2.40×10-5 were obtained in DM and DDM respectively15, for a similar peptide at pH 7.4 via hetero-FRET measurements.
chain lengths from 10-carbon to 16-carbon. Stronger association were observed in detergents with maltose heads than in those with zwitterion heads, while the effects of alkyl chain lengths could not be easily generalized. Similar trends were observed for GpA in various detergents15, despite polarity differences between the two peptide sequences. M2TM was also reported to have tighter association in OG than in DPC via AUC experiments16. Addition of drug resulted in decreasing FP for M2TM, but no effect for drugresistant S31N (Figure 2B), consistent with previous results. The lack of oligomer plateau precluded quantification of M2TM association. In micelles, the apparent energy of membrane protein association is dependent on the detergent concentration. We were able to observe the transition towards the oligomer plateau in 0.5 mM DDM (Figure 3), consistent with the previous observation that GPA association became tighter with decreasing concentration of DDM15. While both M2TM and S31N were best fitted by a monomer-trimer model, the model curve deviated from the raw data for S31N, hinting the existence of equilibriums more complex than a two-state model. Sedimentation equilibrium experiments were carried out for the NBD-labeled M2TM (Figure 4). This peptide appeared to exist in a monomer-dimer-tetramer equilibrium (supporting information) in the concentration range used for FP assay. The dimer species was not observed in previous sedimentation equilibrium experiments with label-free M2TM16-18. This change in the association equilibrium was correctly reflected in the homo-FRET FP curves, while the monomer-trimer model is the result of limitations in fitting capacity. Stable dimer was also detected for spin-labeled M2TM in the lipid bilayer via DEER measurements19. It’s possible that NBDlabeling stabilized a dimer species for the small peptide, and this effect may go away when a larger protein is used.
FIGURE 1. GpA serial dilution results can be fitted well to the monomer-dimer equilibrium model, while GpAmut remained in the monomer state throughout. The monomer-trimer model didn’t converge on any acceptable fit.
FIGURE 3. The best fits for both M2TM (A) and S31N (B) in 0.5 mM DDM is a monomer-trimer equilibrium model, but the fitting for S31N is still not ideal.
Similar FP curves was obtained for M2TM (Figure 2A). The oligomer plateau was not observed, indicating weaker association of M2TM compared to GpA. The monomer FP values can be obtained directly from the curves for most detergents. FP values at higher concentrations were normalized with the corresponding FP value at the lowest concentration in order to compare data from different detergents. Three classes of detergents were tested, with head groups of maltose, phosphocholine or sulfobetaine, and alkyl
FIGURE 4. Optimal fit for SE data of NBD-labeled M2TM is concentration-dependent: monomer-tetramer equilibrium for intermediate concentration (A, 1:120), and monomer-dimer for low concentration (B, 1:250) (Table S5).
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FIGURE 2. M2TM also exhibited monomer-oligomer equilibrium, with the association tightest in detergents with the maltose head-group (A). In DPC, amt bound to M2TM and promoted its association, while no FP change was observed for S31N (B).
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The Kd value obtained from the monomer-trimer fit of our FP data was 2.39×10-7. While monomer-trimer fit to SE data generated a Keq value of 7.43×107 M-2 for the full dataset (Table S5), or 1.15×109 M-2 for unlabeled M2TM from previous publication18, both were carried out in DPC. The relationship between Kd and Keq for a monomer-trimer equilibrium in any detergent solution is described by the following equation, where [M] and [T] are the absolute concentrations of monomer and trimer respectively: 1 = ( ) = × 1 = × FP measurements were carried out in 0.5 mM DDM, which should confer much tighter association compared to the 3.6 to 15 mM DPC used in SE experiments. From the above equation, Keq in 0.5 mM DDM was calculated to be 1.67×1013 M-2, much larger than the Keq values obtained in DPC as expected. Although less information-rich compared to AUC or NMR, our homo-FRET assay is easy to implement and compatible with fast screening. It’s also advantageous for self-association studies compared to conventional FRET or quenching methods, due to easier sample preparation and assay setup. Proteins can either be fused with GFP which has been extensively used to evaluate membrane protein expression levels20, or labeled with strategically placed Cys residue and fluorophores modified with thiol reactive groups. Well-characterized transmembrane peptides, known to form parallel helical-bundles, were chosen for proof of concept here. Antiparallel associations should show similar behaviors, albeit with smaller ranges of FP reduction, as the R0 of fluorophores (2.8 nm for NBD, 5 nm for GFP) is on par with the width of the lipid bilayer (3 nm). For any protein existing in a two-state selfassociation equilibrium, we can now quantify both association states and constants quickly. While for proteins with more complicated equilibria, this method will still be useful when used to screen a large number of experimental conditions. In addition, when compound binding influences membrane protein association, it may be convenient to adapt the assay for quick discovery of novel ligands.
ASSOCIATED CONTENT Supporting Information Detailed materials and methods for peptide synthesis, sample preparation, FP measurements, sedimentation equilibrium, and data analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] Funding Sources We thank the National Natural Science Foundation of China (81402902) and Soochow University for the financial support.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We thank Dr. Manasi Bhate for assistance with the sedimentation equilibrium experiments.
ABBREVIATIONS
DM, decyl-β-D-maltopyranoside; DDM, n-dodecyl-β-Dmaltopyranoside; DPC, n-dodecylphosphocholine; TPC, ntetradecylphosphocholine; HPC, n-hexadecylphosphocholine; TDP, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; HDP, n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.
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Biochemistry
For Table of Contents Only Title: Quantification of Membrane Protein Self-association with a High-throughput Compatible Fluorescence Assay
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Authors: Junbei Li and Xiaoyan J. Qiu
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