Watching Three-Dimensional Movements of Single Membrane


Watching Three-Dimensional Movements of Single Membrane...

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Watching three dimensional movements of single membrane proteins in lipid bilayers Li Ma, Ying Li, Jianbing Ma, Shuxin Hu, and Ming Li Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00253 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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Biochemistry

Watching three dimensional movements of single membrane proteins in lipid bilayers Li Ma†,‡, Ying Li†,‡, Jianbing Ma†,‡, Shuxin Hu†,‡*, and Ming Li†,‡* †

Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft

Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China ‡

School of Physical Sciences, University of Chinese Academyof Sciences, Beijing 100049,

China *To whom correspondence should be addressed. Mailing address: Institute of Physics, Chinese Academy of Sciences, P. O. Box 603, Beijing 100190, China Phone number: 86-10-82649058. Fax number: 86-10-82640224. E-mail: [email protected]; [email protected].

Keywords: Single molecule, Surface-induced fluorescence attenuation, 3D movements of membrane proteins, Membrane-protein interaction

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ABSTRACT: It is challenging to assess protein-membrane interactions because of the lack of appropriate tools to detect position changes of single proteins in ~4 nm range of biological membranes. We developed an assay recently termed surface-induced fluorescence attenuation (SIFA), and it is able to track both vertical and lateral dynamic motion of single labeled membrane proteins in supported lipid bilayers. Similar to FRET principle, SIFA takes advantages of the energy transfer from a fluorophore to a light-absorbing surface to determine the distance of 2-8 nm far from surface By labeling a protein with a proper fluorophore and using graphene oxide as a two-dimensional quencher, we showed that SIFA is capable of monitoring three-dimensional movements of the fluorophore-labeled protein not only inside but also above the lipid bilayer atop the graphene oxide. Our data show that SIFA is a well-suited method to study the interplay between proteins and membranes.

Introduction Membrane proteins regulate various cellular activities and play vital roles in every living cell.1-4 To determine the dynamic behavior of membrane protein is key to understand their biological functions.5 In the past decades, various techniques have been used to study protein-membrane interactions, and have made significant progress, nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, for example. However, well-suited methods essential for mechanistic understanding of the membrane proteins are still lacking.6-8 Sum frequency generation (SFG) vibrational spectroscopy is able to investigate structure and orientation of proteins in lipid bilayers, but it does not yield information about the insertion depth of the molecules.9, 10 Brominated lipids fluorescence quenching method has high resolution, but the method cannot tell in which leaflet of the lipid bilayer the dye is located if both leaflets are

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Biochemistry

brominated.11 Moreover, these techniques yield only time- and ensemble-averaged properties of the molecules of interest. It is still difficult to get detailed dynamic information of the protein around membrane especially when diffusion and stochasticity make protein behavior more complex. Single-molecule techniques have been proven powerful in addressing questions about the interplay between proteins and membranes, providing unique ways to investigate the kinetics of multi-component systems. For example, by using optical tweezers, atomic force microscope (AFM) and magnetic traps, researchers measured the force, affinity, and kinetics associated with proteins and lipid bilayers.12, 13 AFM was also used to show the structure of oligomeric and polymeric assemblies on top of membranes.14,

15

Moreover, single-molecule fluorescence

imaging has been widely used to analyze the assembly process and the membrane bound states of proteins through single-particle tracking and stoichiometry evaluation of each particle.16, 17 Method using environment-sensitive dyes, whose color and brightness is sensitive to the microenvironment, can distinguish whether the environment of the dyes is hydrophilic or hydrophobic.18-20 Taking advantages of high spatial resolution (< 1 nm),21 single-molecule FRET has been successfully used to reveal ion channel structures and dynamics.22, 23 It works well when both the donor and the acceptor are pre-specified.21 However, the donor and the acceptor might become too far to yield FRET signals when the proteins undergo diffusive motions. Moreover, in most studies on membrane proteins, one is just interested in the position of the proteins in the reference frame of the membrane. The movement of a protein in the direction parallel to the membrane is usually concomitant with the movement perpendicular to the membrane because of the fluidity of the membrane. FRET however does not distinguish the two kinds of movements. We recently developed a method that we termed surface-induced

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fluorescence attenuation (SIFA) to track both vertical and lateral kinetics of singly-labeled proteins in supported lipid bilayers.24 It is basically a point-to-plane distance indicator. Here, by labeling the proteins with fluorophores and using graphene oxide as the absorber, we showed that SIFA is able to monitor three dimensional movements of the proteins not only inside but also above the lipid bilayers. SIFA is therefore a powerful tool to study the structural dynamics of membrane proteins. Materials and Methods Fluorescence Microscopy. To obtain small enough background noise, a total internal reflection fluorescence (TIRF) microscope was used. . In order to detect hundreds of single-molecules simultaneously, , two-dimensional detectors such as a charge-coupled device (CCD) camera (e.g., EMCCD, Andor Technology iXon 897) was used.21 A laser (e.g., 532 nm solid state laser (Changchun New Industries Optoelectronics Tech. Co., Ltd.)) with polarization parallel to the surface was used as the excitation source to have high signal to noise ratio. Emission filters are needed to select the emission light (e.g., Semrock 593/40). Cleaning slides and coverslips. Two holes with 1 mm diameter were drilled in the quartz slide, which were used as the inlet and outlet of the chamber. The quartz slide and coverslip were sonicated (250 W) in acetone and methanol for 30 min, respectively. Each time after ultrasonic cleaning, substrates were rinsed thoroughly with deionized (DI) water (>18.2 MΩ•cm) and dried by nitrogen. The slide and coverslip were then cleaned using piranha solution (7:3 H2SO4: H2O2) for 1 hour at 95 °C and washed with DI water and dried by nitrogen. The slides were treated with a plasma cleaner (e.g., PDC-32G-2, Harrick Plasma) with oxygen plasma at the high level for 10

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Biochemistry

min. The chemical reagents were analytical-regent grade (from Beijing Chemical Reagents Company) and used as received without any purification. Deposition of monolayer graphene oxide on the cleaned quartz slides. Langmuir-Blodgett (LB) techniques were used to deposit the single-layered graphene oxide (GO) on the fresh plasma-treated quartz, which was initially prepared by a modified Hummers method.

25,26

The

LB trough was carefully cleaned with chloroform and then filled with DI water. A DI/methanol mixture was used to dissolve the GO solution. A glass syringe was used to slowly spread (100 µL / min) the GO solution (total 8-12 mL) onto the subphase surface of the LB trough. GO film was prepared by compressing barriers at a speed of 20 cm2/min. The surface pressure was monitored using a tension meter. The quartz slide was vertically dipped into the trough and slowly pulled up at a speed of 2 mm/min. GO-covered slides were dried in a vacuum oven at 85 °C for 2 h to enhance the adhesion. Preparation of the sample chamber. The chamber was made by sandwiching a GO-covered quartz slide and a glass coverslip using double-sided tape (e.g., 50 µm thick, 3M Corporation) and then sealed with silicone to make the flow channel. Two thin clean tubes were connected with the inlet and the outlet respectively. A syringe was connected to the outlet tube for extracting. The chamber was assembled immediately before use. GO modified with PEG. A PEG cushion layer is used to increase ~1 nm distance between the bilayer and the graphene oxide surface. Mixture of Aminopyrene (AP, from Sigma-Aldrich) and N-hydroxysuccinimide derivative of a poly-ethylene glycol (NHS-PEG, Average molecular weight 5000, Sigma-Aldrich) was made with the final concentration of NHS-PEG at 2 mM (AP/NHS-PEG, 1/10, mol/mol).The solution was shaken at room temperature for 2 h at 300 rpm

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to make sure chemicals dissolve completely. 200 µL product was injected slowly (50 µL/min) into the fluidic sample chamber and incubated for 3 h. 2 mL deionized water was then injected to wash away the extra solution, and then followed by 400 µL HEPES buffer (20 mM HEPES, 100 mM NaCl, pH = 7.4). Proteins and labeling. DNA constructs for bacterial expression of recombinant full-length mouse Bid with an N-terminal His6 tag and mouse caspase 8 were obtained as described.27 For site-specific fluorescence labeling, Bid cysteine mutants (E80C, L166C or N181C) were made respectively based on the cDNA with cysteine-null mutation (C30A and C126A) by site-directed mutagenesis. The proteins were expressed in Escherichia coli BL21 and purified as described previously.28 The cleaved Bid (cBid) was obtained by adding caspase 8 to the full-length Bid and incubating for 12h at 4°C. Then each cysteine mutant of cBid was labeled by adding the fluorescence dye tetramethylrhodamine-5-maleimide (Sigma) in the molecule ratio of 1:5 (100 µM dye to 20 µM cBid) and incubated at 30°C for 2 h in the dark. Uncombined dyes were removed through dialysis against HEPES buffer (20 mM HEPES, pH 7.4, 200 mM NaCl and 5% glycerol). The human antimicrobial peptide LL-37 was synthesized by China Peptides Co. Ltd. (Shanghai China), whose N-terminus was labeled with rhodamine. Liposome preparation and formation of supported lipid bilayers (SLBs). All lipids (dioleoyl-phosphatidylcholine

(DOPC),

dioleoyl-phosphate

(DOPA)

and

dimyristoyl-

phosphatidylglycerol (DMPG)) were purchased from Avanti Polar Lipids. Specific lipid mixtures were dissolved in chloroform and methanol (2:1, V/V). The solvent was dried under nitrogen flux and then subjected to vacuum for more than 1 h to remove the residual organic solvent. The lipid mixtures were rehydrated using HEPES buffer (20 mM HEPES, 100 mM NaCl, pH = 7.4) to a final concentration of 2 mg/ml. The suspension was then vortexed and incubated

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Biochemistry

for 1 h. At last, the suspension was bath sonicated until obtaining clear transparent solution (small unilamellar vesicles). The suspension was stored at 4°C before use. The small unilamellar vesicles solutions were injected into the chamber and incubated with PEG modified GO at 37°C overnight. The non-fused vesicles were washed away using 2 ml HEPES buffer (20 mM HEPES, 100 mM NaCl, pH = 7.4).

RESULTS Overview of the SIFA approach. The principle of SIFA is similar to FRET, which is the energy transfer from a donor fluorophore to an acceptor through induced dipole-dipole interactions.21, 29, 30 The energy transfer efficiency of FRET follows an equation,  = 1/[1 +  ⁄ ], where is the distance between the donor and the acceptor and is the distance at which half of the energy is transferred.21,31,32 FRET has proven to be a powerful spectroscopic technique for measuring distance changes in the range from about 3 to 8 nm (Figure 1A).33 However, when applied to lipid membranes, it fails to distinguish the movement of the fluorophore along the direction perpendicular to the membrane surface from that parallel to the membrane surface. SIFA does not has such limitation (Figure 1B).34 It is basically the fluorescent energy transfer from a point to a plane such that the movement parallel to the membrane surface is integrated out. The quenching efficiency of SIFA follows the equation,  = 1/[1 +  ⁄  ] , where  is the fluorophore-to-surface distance and  is the characteristic distance at which half of the energy is transferred.35-37 For studying proteins in lipid bilayers, a good choice of  is approximately 4 nm (Figure 1C). We chose GO as the acceptor, which is an excellent quencher for adsorbed fluorophores with a quenching efficiency approaching 100% (Figure 1D).38-41 In general,  can be calculated according to the overlap of

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the spectra of the donor and the GO layer.35,

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It can also be determined by measuring the

fluorescent attenuation of a fluorophore atop a lipid bilayer of known thickness. In this way, we estimated  to be about 4.3 (± 0.5) nm for the GO used in our work. According to the quenching efficiency  , we can get  ⁄ = 1   =  ⁄  ⁄[1 +  ⁄  ] , where  means the fluorophore intensity in the GO system and  means the intensity at the same circumstances without GO. Like FRET, we could easily calculate the fluorophore-to-GO distance, , through the recorded fluorophore intensity.24,29

Figure 1. The principle of SIFA. (A) Schematic of FRET. (B) Schematic of SIFA. (C) Experimental setup of SIFA (left) and the SIFA efficiencies for various  (right). (D) The GO sheets deposited on quartz (left) quench strongly fluorophores on them (right).

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Biochemistry

Three dimensional movements of a membrane protein in a lipid bilayer. SIFA measures the fluorescent intensity of a fluorophore-labeled protein to give information about its z-coordinate in a solid-supported lipid bilayer. By nature, SIFA can also provide information about the xycoordinates of the protein trapped in the membrane by simply analyzing the images. Here we applied SIFA to monitor three dimensional movements of the pro-apoptosis protein Bid in the lipid membrane (Figure 2). Bid is a member of the Bcl-2 (B-cell lymphoma 2) family proteins that are involved in the mitochondrial pathway of apoptosis.20, 42 During apoptosis, it is cleaved by caspase-8 to two fragments (p7 and p15) in response to death stimuli. As a result, Bid is activated and named as cBid, whose two fragments remain together through hydrophobic interactions. Upon membrane binding, the two fragments separate. The first fragment p7 containing two helices is left, and the second fragment p15 named tBid binds and inserts into the membrane to induce the apoptosis. The tBid (C-terminal p15) was labeled with tetramethylrhodamine-5-maleimide at the residue 166. The lipid bilayer is mimicking lysosomal membranes with the phospholipid composition of DOPA:DOPC=1:4. The supported lipid bilayer was produced by direct liposome fusion on the top of GO layer modified with PEG. Figure 2 shows a typical 3D trace (left) and the corresponding fluorescence time course (right) of a tBid molecule labeled at site 166. The behavior of the protein can be described as follows: (i) The protein landed on the membrane at 0.63 s to give the fluorescence signal; (ii) It then diffused on the membrane with a fluorescence intensity of about 60%  in the time interval between 0.66 s and 1.29 s; (iii) The center of the protein (site 166) inserted into the membrane at 1.32 s so that the fluorescence intensity drops to about 25%  ; (iv) The protein diffused two dimensionally in the middle of the membrane from 1.32 s to 2.67 s; And finally, (v) The fluorophore was photobleached at the time 2.70 s.

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Figure 2. 3D detection of a site of interest of a protein. The 3D trace of the fluorophore is shown in the left panel and the corresponding fluorescence time course is shown in the right panel. Details about the dynamics of the protein are in the text. Positions of sites of interest on and above a lipid bilayer. Although SIFA was originally developed to extract the information of insertion depth of membrane proteins in lipid bilayers24, we found that it can do more than that, specifically, it can tell whether the dye is above the membrane surface with high precision. Previously, using environment sensitive dye method researchers can tell whether a dye is in hydrophilic or hydrophobic environment, which can induce weather the dye is in solution or inside the membrane.20 Here we demonstrate the feasibility of SIFA to measure the precise position of a fluorophore around the surface of a lipid bilayer. To this end, we compare the fluorophores at the residues 80 and 181 of tBid. Figure 3A and 3C show the fluorescence intensity traces obtained from tBid labeled at residue 181 and 80, respectively.  and  are obtained from tBid on quartz-supported and a GO-PEG-supported lipid bilayer, respectively. The ratio of  to  for the tBid labeled at residue 181 in Figure 3A is 70 (±4) %, indicating that the residue 181 is on the membrane surface, consistent with previous reports.6, 7 In contrast, as shown in Figure 3C, the ratio of  to  for the residue 80 is 98 (±4) %.

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Biochemistry

It reveals that the residue 80 is exposed in the solution away from the membrane. Previous studies suggest that the BH3 domain of tBid, where the residue 80 is located, binds and activates BAX to induce pores in mitochondrial outer membranes.43, 44 We propose that the conformation of tBid with its BH3 domain exposed to water is likely a prerequisite for effective tBid-BAX interactions.45-47 This work reveals SIFA’s ability to explore the relative position of proteins to membranes at the single molecular level, which can widely be applied to study proteinmembrane interactions.29

Figure 3. Comparison of the positions of the residues 181 and 80 of tBid on lipid bilayers. (A) Typical fluorescent traces of 181-labeled tBid on a GO-PEG-supported bilayer (red line) and a quartz-supported bilayer (grey line). (B) The corresponding probability distribution of the fluorescence intensities. (C) Typical fluorescent traces of 80-labeled tBid on a GO-PEGsupported bilayer (red line) and a quartz-supported bilayer (grey line). (D) The corresponding probability distribution of the fluorescence intensities. The fluorescent signals were observed upon landing of the fluorophores on the membrane and disappeared when they were photobleached. The statistics were obtained from over 250 traces.

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Probing the penetration depth of a single molecule in oligomer. Oligomerization of membrane proteins is involved in various cellular activities, such as apoptosis, signal transduction, and immunomodulation.15,16,48,49 Previous studies on membrane protein oligomerization focused on the diffusion coefficients and the stoichiometry of the oligomers in the membrane.17, 49 However, the measurements are not able to yield the penetration depth of a single molecule within the oligomers. Here we show that SIFA is capable of providing such information. LL-37 is a 37-residue cationic peptide with a broad spectrum of antimicrobial activity and is involved in immunomodulation, angiogenesis and wound healing.50, 51 Previous reports demonstrated that LL-37 induces pores in lipid bilayers.51 Here we examine the position of the N-terminus of LL-37 in the pores formed in the lipid bilayer (Figure 4). After incubating the fluorophore-labeled LL-37 with the membrane for 10 minutes, we took continuous images to photo-bleach the fluorophores in the pores. Many pores displayed step-by-step photo-bleaching. The photo-bleaching steps are uniform when the lipid bilayer is deposited on a native quartz surface (Figure 4A). In contrast, the steps are not uniform and distribute around three intensities when the lipid bilayer is supported by the GO layer (Figure 4C). Interestingly, when we mixed a small amount of labeled LL-37 with unlabeled LL-37 to ensure that each pore contained only one labeled LL-37,24 we found that the fluorescent intensity fluctuated between three states, of which the relative intensities  ⁄ are