Atomic Force Microscopy Topographs Research Articles

High-Resolution AFM of Membrane Proteins Directly Incorporated at High Density in Planar Lipid Bilayer

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl- β-maltoside or dodecyl- β-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20°C and at 4°C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.

An Approach To Prepare Membrane Proteins for Single‐Molecule Imaging

Membrane proteins are under represented in the database of high-resolution structures obtained from X-ray crystallographic and NMR spectroscopic methods. [1] Furthermore, they often form large and sometimes transient supramolecular complexes. Alternative approaches such as cryo-electron microscopy (EM) and Atomic force microscopy (AFM) are essential tools that are able to provide complementary information on the structure–function relationship of membrane proteins embedded in the lipid membrane. Two approaches increase the resolution of these methods, 2D crystallization and single-particle reconstruction. In EM, crystals provide electron diffraction data which can increase resolution, whereas single-particle averaging can be used for proteins > 200 kDa. High-resolution AFM topographs can reveal structural details of single native membrane proteins but, as a prerequisite, the proteins must be adsorbed to atomically flat mica and densely packed in a membrane to restrict their lateral mobility. [2] Although averaging of single particles shows their common structures, selected examples can be used to characterize structural flexibility, variability, and conformational changes. [3] Many examples of AFM analysis of self-assembled monolayers on gold have been published, [4] and thiolipids have been developed to create membrane mimetic surfaces on gold. [5–7] However, these have not yet been combined as a means to image membrane

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of ≈1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of ≈67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of ≈3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of ≈4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.

The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory.

The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically assembled subunits. The c15-mer reported here is the largest c ring that is isolated and does not show the classical c-ring mismatch to the three-fold symmetry of the F1 domain.

Analyzing focal adhesion structure by atomic force microscopy

Atomic force microscopy (AFM) can produce high-resolution topographic images of biological samples in physiologically relevant environments and is therefore well suited for the imaging of cellular surfaces. In this work we have investigated focal adhesion complexes by combined fluorescence microscopy and AFM. To generate high-resolution AFM topographs of focal adhesions, REF52 (rat embryo fibroblast) cells expressing YFP-paxillin as a marker for focal adhesions were de-roofed and paxillin-positive focal adhesions subsequently imaged by AFM. The improved resolution of the AFM topographs complemented the optical images and offered ultrastructural insight into the architecture of focal adhesions. Focal adhesions had a corrugated dorsal surface formed by microfilament bundles spaced 127+/-50 nm (mean+/-s.d.) apart and protruding 118+/-26 nm over the substratum. Within focal adhesions microfilaments were sometimes branched and arranged in horizontal layers separated by 10 to 20 nm. From the AFM topographs focal adhesion volumes could be estimated and were found to range from 0.05 to 0.50 microm(3). Furthermore, the AFM topographs show that focal adhesion height increases towards the stress-fiber-associated end at an angle of about 3 degrees . Finally, by correlating AFM height information with fluorescence intensities of YFP-paxillin and F-actin staining, we show that the localization of paxillin is restricted to the ventral half of focal adhesions, whereas F-actin-containing microfilaments reside predominantly in the membrane-distal half.

Structural evidence for a constant c11 ring stoichiometry in the sodium F‐ATP synthase

The Na+-dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria.

Actin microridges characterized by laser scanning confocal and atomic force microscopy

We have characterized the cell surface of zebrafish stratified epithelium using a combined approach of light and atomic force microscopy under conditions which simulate wound healing. Microridges rise on average 100 nm above the surface of living epithelial cells, which correlate to bundles of cytochalasin B-insensitive actin filaments. Time-lapse microscopy revealed the bundles to form a highly dynamic network on the cell surface, in which bundles and junctions were severed and annealed on a time scale of minutes. Atomic force microscopy topographs further indicated that actin bundle junctions identified were of two types: overlaps and integrated end to side T- and Y-junctions. The surface bundle network is found only on the topmost cell layer of the explant, and never on individual locomoting cells. Possible functions of these actin bundles include cell compartmentalization of the cell surface, resistance to mechanical stress, and F-actin storage.

Fabrication of avidin single molecular layer on silicon oxide surfaces and formation of tethered lipid bilayer membranes

Single molecular layer of avidin is fabricated on an atomically flat SiO2 surface and characterized by atomic force microscopy (AFM) and infrared reflection absorption spectroscopy. Immobilization of avidin is performed as follows; i) ester-modification of the surface by silane-coupling agent, ii) carboxylation by hydrolysis in HCl and iii) amide bonding between the surface -COOH and -NH2 in avidin molecules. Large dome structures (∼60 nm height) are formed after the ester-modification, but an atomically flat surface is obtained after the hydrolysis reaction. AFM topographs and function-recognizing images show that the each of avidin molecules adsorbs as a single molecule and retains the biotin-binding activity. Formation of a tethered bilayer membrane of a biotinylated phospholipid on the avidin layer is also described. [DOI: 10.1380/ejssnt.2005.237]

Structure of the Dimeric PufX-containing Core Complex of Rhodobacter blasticus by in Situ Atomic Force Microscopy

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

Assembly of collagen into microribbons: effects of pH and electrolytes

Collagen represents the major structural protein of the extracellular matrix. Elucidating the mechanism of its assembly is important for understanding many cell biological and medical processes as well as for tissue engineering and biotechnological approaches. In this work, conditions for the self-assembly of collagen type I molecules on a supporting surface were characterized. By applying hydrodynamic flow, collagen assembled into ultrathin (∼3 nm) highly anisotropic ribbon-like structures coating the entire support. We call these novel collagen structures microribbons. High-resolution atomic force microscopy topographs show that subunits of these microribbons are built by fibrillar structures. The smallest units of these fibrillar structures have cross-sections of ∼3 × 5 nm, consistent with current models of collagen microfibril formation. By varying the pH and electrolyte of the buffer solution during the self-assembly process, the microfibril density and contacts formed within this network could be controlled. Under certain electrolyte compositions the microribbons and microfibers display the characteristic D-periodicity of ∼65 nm observed for much thicker collagen fibrils. In addition to providing insight into the mechanism of collagen assembly, the ultraflat collagen matrices may also offer novel ways to bio-functionalize surfaces.

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is approximately 94 A in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 A, and it sits 21 A above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 A. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.

Observing Membrane Protein Diffusion at Subnanometer Resolution

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.

AFM Characterization of Tilt and Intrinsic Flexibility of Rhodobacter sphaeroides Light Harvesting Complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied.The two-dimensional crystals examined had sizes of up to 5μm and, as revealed by a 10Å cryo electron microscopy projection map, p22121 crystal symmetry. The unit cell had dimensions of a=b=150Å and γ=90°, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10Å. Further, the high vertical resolution of ∼1Å, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1Å on one side and 3.8Å on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2° tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to ∼15Å.

Single Proteins Observed by Atomic Force Microscopy

Here we discuss the experimental approaches that have allowed high resolution atomic force microscopy (AFM) imaging, and review results that show AFM to be of great interest for biologists, AFM allows single proteins to be imaged under physiologically relevant conditions. The exceptional signal-to-noise ratio and resolution of AFM topographs enables the oligomerization state and characteristic substructures of individual proteins to be resolved. Several examples demonstrate the capabilities of AFM to directly observe single proteins, and their conformational changes, to study protein-protein interactions and to follow the assembly of membrane proteins. We consider the AFM techniques that have allowed high resolution imaging, and review results that show AFM to be a powerful method to analyze biological processes at the level of single molecules.

Single Proteins Observed by Atomic Force Microscopy

Here we discuss the experimental approaches that have allowed high resolution atomic force microscopy (AFM) imaging, and review results that show AFM to be of great interest for biologists, AFM allows single proteins to be imaged under physiologically relevant conditions. The exceptional signal-to-noise ratio and resolution of AFM topographs enables the oligomerization state and characteristic substructures of individual proteins to be resolved. Several examples demonstrate the capabilities of AFM to directly observe single proteins, and their conformational changes, to study protein-protein interactions and to follow the assembly of membrane proteins. We consider the AFM techniques that have allowed high resolution imaging, and review results that show AFM to be a powerful method to analyze biological processes at the level of single molecules.

Structural Investigations on Self-Organized Si/SiGe Islands by Grazing Incidence Small Angle X-Ray Scattering

We present a novel technique, based on grazing incidence small angle X-ray scattering (GISAXS), with which information on the shape and the lateral correlation of buried islands can be obtained. The GISAXS measurements were performed on Ge-rich islands grown by molecular beam epitaxy on (001) vicinal substrates with a miscut 2° along the [100] direction. By varying the angle of incidence, GISAXS data sets were obtained for different information depths. These data were analysed quantitatively using a model based on the distorted wave Born approximation. In order to demonstrate the capabilities offered by this technique a 20 period Si/SiGe island multilayer sample was investigated before and after an annealing step at about 750 °C for 80 min. The GISAXS data show that the islands change their shape after annealing. For the top island layer a comparison of the GISAXS data with atomic force microscopy topographs was made.

Reversible loss of crystallinity on photobleaching purple membrane in the presence of hydroxylamine

Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.

Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

Background: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. Results: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5–0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. Conclusions: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.

Charting the Surfaces of the Purple Membrane

The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.

Bulk and surface characterization of a dewetting thin film polymer bilayer

We have monitored the progression of the dewetting of a partially brominated polystyrene (PBrS) thin film on top of a polystyrene (PS) thin film with scanning transmission x-ray microscopy (STXM) as well as photoemission electron microscopy (PEEM). We mapped the projected thickness of each constituent polymer species and the total thickness of the film with STXM, while we determined the surface composition with PEEM. Our data show that the PBrS top layer becomes encapsulated during the later stages of dewetting and that atomic force microscopy topographs cannot be utilized to determine the contact angle between PBrS and PS.