Observing Membrane Protein Diffusion at Subnanometer Resolution

doi:10.1016/S0022-2836(03)00206-7

J. Mol. Biol. (2003) 327, 925–930

COMMUNICATION

Observing Membrane Protein Diffusion at Subnanometer Resolution Daniel J. Mu¨ller1,2*, Andreas Engel3, Ulrich Matthey4, Thomas Meier4 Peter Dimroth4 and Kitaru Suda4 1 Max-Planck-Institute of Molecular Cell Biology and Genetics, Biotechnology Center, Pfotenhauerstr. 108 01307 Dresden, Germany 2

BIOTEC, Technical University 01062 Dresden, Germany 3

M. E. Mu¨ller Institute Biozentrum, 4056 Basel Switzerland

4 Institute for Microbiology ETH, 8092 Zu¨rich Switzerland

Single sodium-driven rotors from a bacterial ATP synthase were embedded into a lipid membrane and observed in buffer solution at subnanometer resolution using atomic force microscopy (AFM). Time-lapse AFM topographs show the movement of single proteins within the membrane. Subsequent analysis of their individual trajectories, in consideration of the environment surrounding the moving protein, allow principal modes of the protein motion to be distinguished. Within one trajectory, individual proteins can undergo movements assigned to free as well as to obstacled diffusion. The diffusion constants of these two modes of motion are considerably different. Without the structural information about the membrane environment restricting the moving proteins, it would not be possible to reveal insight into these mechanisms. The high-resolution AFM topographs suggest that, in future studies, such data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein – protein arrangements. q 2003 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: atomic-force microscopy; ATP synthase; diffusion; single molecule dynamics; supported membrane

Molecular interactions regulate life with molecular machineries functioning in a heterogeneous manner. Single-molecule techniques provide insight into the individuality of biological macromolecules, their unique function, their reaction pathways, their trajectories and into their molecular interactions.1,2 Complementary to conventional ensemble measurements, these techniques will provide new biological insights and will assist in understanding molecular principles of modern biology. Conventionally, the location, dynamics and function of single biological molecules are detected by fluorescence microscopy. Fluorescence microscopy, used frequently in modern biological research, requires labeling the molecules of particular interest with fluorophores. These chemically fixed labels, however, exhibit the potential to influence the native behavior of the labeled molecule.3,4 Abbreviations used: AFM, atomic-force microscopy; MSD, mean-square displacement. E-mail address of the corresponding author: [email protected]

Chemical reactions limit the lifetime of the fluorophore and, thereby, the visualization process by optical microscopy. Most importantly, molecules other than the labeled ones will not be visualized by optical methods. Multicolor imaging by the use of various fluorophores allows simultaneous imaging of different biological structures. Unfortunately, the number of colors that are detectable simultaneously is presently limited to a few, and the selection of structures that may be observed has to be chosen carefully. As a side-effect, such pre-selection of labeled structures, before optical imaging, will necessarily exclude most other components of, for example, a biological cell. To detect single molecules by light microscopy, the density of the labeling fluorophores should be reasonably diluted to prevent overlapping of their point spread functions and, thereby, enabling their detection and localization as single molecules.4 As a result, optical single-molecule detection allows observation of only very few different structures, which are separated by a few tens of nanometers. Hence, optical single-molecule detection by fluorophores is an excellent method to localize the

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

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Diffusion of Single Membrane Proteins

samples is performed in buffer solution and at ambient temperatures,11 which allows the imaging of living cells12 and single proteins13 at work.14,15 Such experiments can be performed over a timerange of several hours without destruction of individual proteins or disturbing their inherent assembly. We report here, for the first time, that AFM can be applied to observe the diffusion of single membrane proteins at a lateral resolution of , 1 nm and a vertical resolution of 0.1 nm. Imaging loosely packed membrane proteins at subnanometer resolution

Figure 1. Sodium-driven rotors from Ilyobacter tartaricus ATP synthase embedded in a lipid membrane. Individual rotors were orientated with one end towards the membrane surface. While one end of the rotor was plugged by phospholipid headgroups (left inset) the other end had a central depression (right inset). The plugged and unplugged ends exhibited an average outer diameter of 5.4(^0.3) nm and of 5.7(^0.3) nm protruding by 2.5(^0.3) nm and by 1.5(^ 0.3) nm from the lipid surface, respectively.16 Apparently, some rotors formed assemblies while others had no contact to surrounding proteins. Vertical brightness range of topograph, 3 nm. Sodium-driven ATP synthase rotors were isolated from I. tartaricus31 and reconstituted into a POPC bilayer as described.16 The sample was adsorbed onto freshly cleaved mica17 in buffer solution (10 mM Tris – HCl (pH 7.8), 300 mM KCl). To minimize the force interacting locally between the AFM tip and the protein, the electrostatic double-layer force was adjusted by reducing the salt concentration of the buffer solution to 100 mM KCl (10 mM Tris –HCl, pH 7.8).18 The AFM instrument used was a Nanoscope III (Digital Instruments, Santa Barbara, CA) equipped with a 120 mm scanner and oxide-sharpened Si3N4 tips on a cantilever with a nominal spring constant of 0.1 N/m (Olympus, Tokyo, Japan). Topographs recorded in trace and retrace scanning directions using contact mode showed no significant difference. All measurements were carried out at room temperature.

position of one or a few molecules with a high precision of . 10 nm, but it does not allow one to image biological structures at such a resolution. Thus, experiments detecting the movement of membrane proteins or of lipids by fluorescence microscopy derive conclusions on the surrounding membrane structure indirectly from the diffusion behavior of the proteins.5 – 8 In contrast to optical microscopy, the exceptionally high signal-to-noise ratio of the atomic force microscope (AFM)9 allows the observation of single unlabeled macromolecules in a heterogeneous environment.10 AFM imaging of biological

We have reconstituted ion driven rotors of llyobacter tartaricus ATP synthase into a lipid (palmitoyl-oleoyl phosphatidylcholine; POPC) bilayer16 and adsorbed the membranes onto freshly cleaved mica.17 After this, the sample was imaged at room temperature in buffer solution by contact mode AFM. To minimize the interaction between the AFM stylus and the sample, the force applied to the stylus was counterbalanced electrostatically to < 25 pN.18 The AFM topograph revealed single rotors protruding from the lipid bilayer (Figure 1). While some of the rotors were assembled into densely packed regions, other rotors apparently had no directly adjacent proteins. Imaged at higher resolution, individual subunits of the ring-shaped rotors were observed indicating an 11-fold symmetry (Figure 2(A); and see the inset in Figure 1). Each of the subunits is formed by two transmembrane a-helices connected by a polypeptide loop.19 Observing the motion of single membrane proteins Imaging the same membrane area after 90 seconds showed most rotors remained at their location (Figure 2(B)). A few rotors, however, changed their position. At the same time as some rotors disassembled, others assembled. Repeated imaging of the same area (Figure 2(C)) showed the continuous movement of individual proteins. Tracking individual rotors indicated that the molecules started and stopped their movement at any given time, independent of whether they had moved previously. The membrane protein movement observed was independent of the scanning direction of the AFM stylus (horizontal to topograph) and was not correlated to the location and movement of other proteins. Therefore, we conclude that the imaging process did not influence their motion significantly. However, the diffusion coefficient of < 1.04 £ 1025 mm2/second estimated from topographs recorded in time-lapse movies was much lower than expected from peptides embedded in POPC vesicles (3 £ 1022 –8 £ 1022 mm2/ second).20 The following two reasons may explain the observed reduced diffusion constant. Firstly, membrane proteins moving in supported lipid membranes exhibit diffusion coefficients orders of

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Analyzing the modes of motion

Figure 2. Diffusion of single rotors observed at a spatial resolution ,1 nm. (A) AFM topographs showing rotors assembled in the membrane. Subunits of individual rotors were observed directly in the raw data. (B) Same area imaged after 90 seconds. Individual rotors changed their location (outlined red) while others remained at their location (outlined yellow). (C) Repeated imaging of the same area after an additional 90 seconds. While some proteins moving in the previous images interrupted their movement (outlined blue) others continued (outlined red) or began to move (outlined green). The circles dotted in yellow outline proteins, which did not change their location. Vertical brightness range of topograph, 3 nm.

magnitude lower than when measured in freestanding membranes.21,22 When adsorbed to atomically flat mica, the membranes can be assumed to be separated by a water-layer of < 1 nm, which couples frictional forces between membrane protein and support, but still allows proteins to move within the membrane.21 The result of this effect is observed directly by the reduced diffusion coefficient determined from our measurements. Thus, from experiments performed on supported membranes one cannot necessarily expect diffusion coefficient values similar to that measured on native cell membranes. Secondly, because of size constraints, single peptides of the size of a single transmembrane helix tend to show a higher diffusion constant compared to the oligomeric ATP synthase rotor containing 22 helices such as investigated in this work.

Individual rotors can undergo lateral and rotational movements, as apparent from shifts of their relative position (Figure 2). The uncertainty when determining the position of a single rotor was < 1 nm, on average, which is about 30 times smaller than the accuracy of optical single molecule methods to detect molecular diffusion3,4 and about 200– 300 times smaller than the diffraction-limited width of an excellent optic. This positional accuracy combined with the high spatial resolution of the AFM topographs allowed us to visualize the trajectory of individual rotors and to study their collisions with surrounding molecules. Analyzing all trajectories, without applying any further classification procedures, results in a nonlinear diffusion behavior (Figure 3; inset, black data). From the AFM topographs, however, we can principally distinguish between the structural factors that limit the free diffusion of a membrane protein. It becomes clear that apparent transitions among modes of diffusion can be observed directly. Most obviously, assemblies formed by the tight rotor packing restrict the free diffusion of moving rotors. Additionally, it was observed that some of the rotors assembled with other rotors, bringing their free diffusion through the membrane to an end. To characterize the diffusion behavior of the free motion and of the so-called obstacled motion we analyzed 30 trajectories of rotors, which had no apparent contact with the surrounding proteins during their movement (Figure 3; green circles), and 30 trajectories of rotors, the movements of which had been restricted by other rotors or by their assemblies (Figure 3; blue circles). To ensure that rotors moving large distances had no intermittent contact with surrounding proteins, their motions were analyzed only from diluted areas. As expected, the observed mean-square displacements (MSD) and the time lag ðtlag Þ yielded a linear relationship for the free diffusion (Figure 3; inset, green data23) with a diffusion constant of 2.04 £ 1025 mm2/second. From this almost linear behavior, it can be assumed that the movement of these rotors was not affected significantly by non-diffusive motions or by collisions.23 Rotors showing a restricted motion, however, yielded a non-linear relationship (Figure 3; inset, red data). Interestingly, the MSD for a given time lag fluctuated, thereby showing various gradients. However, the gradient was always smaller compared to that of the free diffusion and showed an average diffusion constant of 0.32 £ 1025 mm2/second. As observed directly from the topographs, it follows that the obstacled motion is represented by a mixture of free motion and of the interactions with other membrane proteins. It may be further assumed that the density and the distribution of the proteins influence this effect. To investigate to what extent the imaging parameters of the AFM showed an influence on the

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individual rotors start and end their movement independently from each other, we assume the effect of the scanning process on the rotor movement to be neglectible in this work. Conclusions and biological significance Direct observation of membrane protein motion and membrane structure

Figure 3. Trajectories of the two-dimensional diffusion of single cation-driven rotors in a lipid membrane. Analyzing the AFM topographs, it was possible to distinguish between free motion of rotors that had no apparent contact with surrounding membrane proteins (green circles) and obstacled motion of rotors that had limited free space due to interactions with other proteins (blue circles). Inset, MSD versus time lag calculated for 30 trajectories. The black dots represent all motions, which were not further classified. The green and blue dots present the intermediate steps of the free and obstacled rotor motion, respectively.

rotor movement, we varied the scanning parameters such as scanning speed, feedback and imaging force. It appeared that the rotor movement could not be directed by the scanning stylus. At sufficiently high imaging force, @ 100 pN, membrane and individual proteins became disrupted by the scanning AFM stylus. Similarly, the feedback parameters controlling the tip-sample interaction were crucial to prevent destruction of the proteins and showed the same effect as elevated forces. At excessive scanning speeds, the feedback control is not sufficient and the force interacting between the AFM stylus and the membrane lacked control. Again, the membrane and protein surfaces were disrupted by the scanning stylus. Only if AFM topographs recorded successively from the same membrane surface showed the individual protein structures unchanged, were the imaging parameters (force, scanning speed, feedback control) adjusted optimally to prevent observable structural or functional influence of the sample. Nevertheless, no connection between the diffusion direction of the rotors and the scanning direction of the AFM stylus was observed. It cannot be ruled out, however, that the interaction between the rotors and AFM tip enhances the probability of a rotor to diffuse or initiates the rotor diffusion through the membrane. However, since there was no dependence of the rotor movement on the scanning direction, and since

Most importantly for molecular cell biologists, it has been demonstrated that individual biological molecules can be observed moving through a heterogeneous assembly of membrane proteins. As revealed from AFM topographs, the movement of single rotors was restricted by surrounding single motors and by rotors forming assemblies. Occasionally, the moving rotors reversibly joined either one of the obstacles. On the other hand, individual rotors moved freely through the lipid membrane without showing apparent interactions with other proteins. The observed trajectories suggest that the membrane proteins undergo apparent transitions between these modes of motion (Figure 3; inset, black data). Subsequent analysis of the diffusion behavior revealed the first type of movements to be comparable to a mixture of corralled and obstacled diffusion (Figure 3; inset, blue data). The second type of movement is described best by free diffusion (Figure 3; inset, green data). The diffusion constants measured, however, were orders of magnitude lower than that observed for a free membrane, a behavior explained by the ultrathin water layer (< 1 nm) sandwiched between the membrane and supporting mica surface, which couples the friction between protein and the support. The example given in Figure 2 shows only three consecutive topographs of the same membrane area. Within these images only a few rotors changed their location, most of the other rotors remained at their location. However, observing this and other areas over a much longer time-range indicated that almost all proteins moved. Therefore, we assume that if we could observe the proteins over an even more extended time-range, the individuality of more and less mobile rotors would be smeared out over the time. This hypothesis, however, has to be proven. One other approach to investigate the individual protein mobility would be to observe a membrane exhibiting a greater fluidity (e.g. at enhanced temperatures or different lipid compositions). To do so, however, AFM topographs must be captured within a much shorter time-range. Therefore, we are currently establishing high-resolution imaging using fast-speed AFM in our laboratory (see discussion below). Heading to address fundamental biological questions Driven by their dynamic clustering, lipids, other amphiphilic molecules and membrane proteins

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have been found to form rafts that move within the cell membrane.24 Such rafts are proposed to serve as platforms for the attachment of proteins controlling protein sorting and trafficking through secretory and endocytic pathways. The assembly of membrane proteins into rafts appears to be of significant importance during signal transduction25 and many other biological processes.26 It remains a challenge to understand the molecular mechanisms driving the assembly of rafts. In this context, techniques allowing the observation of the de- and re-assembly of membrane proteins into functional assemblies27 can be seen as a step towards studying the formation of functional membrane protein assemblies. Furthermore, the ability to image individual proteins and of their environment will allow more complex biological systems to be studied and will provide novel insights into the biogenesis of various native membranes, into the interactions between similar or different membrane proteins, and into their formation of supramolecular complexes. Towards higher temporal resolution and better understanding of molecular assemblies Imaged at a spatial resolution of , 1 nm, the molecular arrangement of assembled proteins was resolved in this work. Because of the relatively low temporal resolution of the AFM used, only slow motions of the membrane proteins could be studied in this work. Many biological processes take part on a much faster time-scale. Fast scanning AFM allowing imaging of the biological sample at much greater time resolution28 – 30 will allow observation of the dynamics of biological processes in greater detail. After establishing these fast AFM techniques for high-resolution imaging of single proteins they may be applied to study faster diffusion and assembly processes on supported membranes exhibiting an enhanced mobility. Immobilized labels allowing the measurement of fast protein movements of highly diffusive systems may be required and provided by adsorbing nanogold particles or filamentous structures onto the membranes. With these or similar approaches, a combination of experimentally observed protein movements, influenced by various biochemical parameters (e.g. pH, electrolyte, lipid composition, temperature, trajectories), and of molecular modeling of the complexity of the protein arrangement will promote the understanding of protein assembly and will provide unique insights into mechanisms that drive intermolecular interactions in cellular membranes.

Acknowledgements We thank Kurt Anderson, Joe Howard, Kate Poole, Kai Simons, and Winchil Vaz for critical

reading of the manuscript. This work was supported by the Maurice E. Mu¨ller and Swiss National Foundation (SNF), by the European Community (EFRE) and the Sa¨chsische Ministerium fu¨r Wissenschaft und Kunst (SMWK), Germany.

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Edited by W. Baumeister (Received 14 October 2002; received in revised form 6 January 2003; accepted 6 January 2003)