SPM Applications in Biology

     Proteins play fundamental role in structuring and vital functions of all living creatures. This wide class of biomolecules includes well-known names such as albumin, hemoglobin, insulin. Atomic Force Microscopy from its very beginning contributed significantly in understanding the peculiarities of proteins functioning as well as provided extra information about their structure and properties.

     Atomic Force Microscopy usefully employed in exploring protein adsorption onto solid surfaces along with radiolabeling, fluorescence spectroscopy, ellipsometry and other methods. It is quite important in investigation of implant biocompatibility, in vitro cell growth, membrane fouling, protein purification and biosensor design. The behavior of proteins at surface defect sites is of interest, as such defects may provide a means of immobilizing biological molecules for detection purposes [1086]. Protein-covered surfaces may be also useful for the catalysis of biological reactions.

     Y. F. Dufrene at al. [677, 1527] investigated the organization of collagen adsorbed onto polymer substrata. Combining XPS and radiolabeling they proposed a quantitative description of the layer on the basis of a simple geometric model. AFM allowed to confirm this organization by direct observation of the continuous or discontinuous character of the adsorbed layer and provided novel information by revealing topographic features at supramolecular scale (fibrillar structures).

     A.P. Quist at al. [804] studied the adsorption of albumin (HSA) and tripsin molecules on mica surfaces using SFM. The observed hillocks indicate that molecules adsorb partly as aggregates and partly as isolated single molecules. A qualitative estimate of the profiles of the adsorbed molecules can be obtained, giving vivid information on the conformation and domain structure of adsorbed molecules. Individual molecules were resolved. By the opinions of the authors, it is very exciting that the structure and conformation of individual molecules can be observed with TM-SFM, making it a powerful tool for biological research.

     P. Kernen at al. [869] investigated aggregations of the largest light-harvesting pigment-protein complex of Photosystem II (SHC II) deposited on glass using Langmuir-Blodget films technique. Formation of Langmuir-Blodget films with incorporated biomolecules of interest is a common way in preparing flat mono- or multilayer species for measurements with various methods including AFM. Direct observation of structural organizations in these films helps us to understand specific interactions between molecules within the layer. SHC II is an antenna protein in higher plants comprising almost half of the total pool of the main photosynthetic accessory pigment chlorophylls. Ring-like structures formed in monocomponent protein layers as well as in mixed protein-lipid films were revealed using AFM. It is suggested that LHC II organizes as round-shaped circles with internal diameters of 150-250 Å and external diameters of 300-500 Å.

     Epand at al. [696] first applied atomic force microscopy to a study of the properties of the hemagglutinin (HA) protein of influenza virus. Association of two different forms of the ectodomain of this protein at supported lipid bilayer interfaces as a function of pH and incubation time was explored. These are bromelaincleaved hemagglutinin (BHA), corresponding to the full ectodomain of the HA protein, and FHA2, the 127 amino acid N-terminal fragment of the HA2 subunit of the hemagglutinin protein. The results provided direct evidence of different protein aggregation phenomena at model lipid surfaces for the BHA and FHA2 fragments of the influenza HA, that may be relevant to their function. The results presented in this paper are the first example of in situ imaging of the ectodomain of a viral envelope protein allowing characterization of the real-time selfassembly of a membrane fusion protein.

     Nondestructive character of Atomic Force Microscopy and possibility of operation in nearly physiological conditions prompted studies of lachrymal deposits on Soft Contact Lens (SCL) that are mainly composed of proteins. J. Baguet at al. [445] suppose AFM is a new exceptional tool for exploring biomaterials and biomolecular-surface interactions by extending the atomic resolution of the scanning tunnelling microscope to non-conducting materials. The use of Scanning Electron Microcopy in such a case faces several disadvantages since lens preparation affects the structure and the surface of the unworn and worn lenses, some deposits are artefactual and the damaging electron beam causes SCL destruction. For proteins identification combination of AFM and a sodium dodecil sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of extracted SCL deposits were performed in parallel fashion. Thus, new and unique information on SCL deposits from contacting lachrymal component shows that adsorption on surfaces during continuous wear of the soft contact lenses is a two-step mechanism. First, a uniform coating, probably composed of proteins and mucosubstances, covers the surface. Second, structured deposits appear on the lens surface and quickly form an additional layer over the first protein coating. The images clearly show the evolution of the size and structure of these deposits.

     Large attention in scientific world also attracts growth of proteins from solutions in crystalline form [118]. During a last decade a number of in situ AFM studies were performed on lysozyme [119, 546, 1087, 1088, 1089], canavalin [235, 597, 759, 1090, 1092], thaumatin [546, 755, 1091, 1092], a-amilase [300], catalase [222]. Studying the processes of macromolecular crystallization helps to understand better growth kinetics and nucleation mechanisms in crystal growth at all. For instance, investigation of growth behavior of porcine pancreatic a-amylase at defined supersaturation, performed by J.P. Astier at al. [300], revealed that at high supersaturation (b=1.6) 2-D nucleation is to be the dominating growth mechanism, whereas at lower supersaturation (b=1.3) the growth process appears to be defect controlled (spiral growth). The analysis of step heights on 2-D nucleation islands (monomolecular protein layers) and growth steps (two molecules in height) in combination with results from light scattering experiments suggest that a single protein molecule is the basic growth unit.

     Although similar and higher resolution can be obtained by electron microscopy and X-ray crystallography, the excellent signal-to-noise ratio of AFM topographs allows the direct imaging of native proteins [309, 522, 1094-1098] and their substructures to a resolution of about 0.5 nm [1099]. AFM enables conformational changes of single proteins and of their assemblies to be observed directly [1501-1504]. Furthermore, conformational changes can be induced in a controlled manner to identify flexible protein structures [1095, 1098, 1505].

     The plasma membrane of the cell accommodates diverse membrane proteins, including integral membrane proteins such as receptors, ion channels and transporters, as well as certain antigens that are peripherally associated with the membrane. Because of their important roles in cell growth, differentiation and cell-cell signaling, the structures of the plasma membrane and proteins associated with it have attracted wide attention and have been extensively investigated. During last twelve years the study of native membrane proteins evolved from measuring AFM topography of protein layer to a single-molecule force spectroscopy [381, 1503, 1505, 1517-1524]. In situ AFM investigations of protein-lipid interactions were also performed [1081-1085]. Continuous progress in AFM apparatus, measuring technique and sample preparation apparently can be seen for one of the most popular object of protein nature ever has been imaged with AFM - bacteriorhodopsin (BR) covering the purple membrane (PM). This protein acts as a light driven proton pump to produce a finite difference in the proton concentration between the inside and outside of the cell membrane [256]. As summarized by Müller at al. [381], trimeric BR molecules arrange in a trigonal lattice of 6.2?±0.2 nm side length. Power spectra of observed structure suggest lateral resolution as low as 0,45 nm. Such excellent spatial resolution as well as extrasensitivity at low cantilever loading ranged from 100 nN to 300 nN allowed to investigate the major conformations of BR surfaces and to map the variability and the flexibility of individual polypeptide loops connecting transmembrane K-helices of BR. It was revealed that full conformation of trimer is accomplished when loading force rises from 100 pN to 200 pN. Application of force up to 300 pN results in a deformation of the peripheral protrusions of the trimer and structural information of these areas is lost. Detailed analysis of images obtained gave rise to differentiate six K-helices of the protein according to their flexibility under load applied. Comparison of AFM data and atomic models of BR (to date six model were offered) derived from electron and X-ray diffraction experiments are presented. There is an excellent correspondence between the surface loops of the BR model and the AFM envelope. Standard deviation maps of the height measured by AFM correspond well with the relative distribution of B-factors of the atomic models as well as the coordinate variance between the models. S.D. maps allows for revealing the elasticity of single polypeptide loops. In contrast to electron and X-ray crystallography methods, the AFM can be used to image surface structures of BR in buffer solution and at room temperature similar to their physiological environment. All these evidence that the AFM not only fulfills the prerequisites to directly monitor function related conformational changes of biological macromolecules [427, 1093, 1502, 1504] but can also characterize dynamic aspects of protein structures, such as their flexibility and variability.

     In single molecule force-spectroscopy experiments, the protein complexes are tethered to both support and AFM tip to measure their cohesion when tip and support are moved apart. This technique has been employed to measure forces between pairs of interacting biological molecules [789, 790, 794, 795, 797, 1509] and forces required for the unfolding of titin domains [1512-1514]. Protein complexes were imaged before and after the removal of individual subunits using the AFM tip as a dissecting nanotool [1096]. Based on these results, the single molecule imaging and single molecule force-spectroscopy capabilities of the AFM have been combined to provide novel insights into the inter- and intramolecular interactions of proteins [1515, 1516]. Applied to membrane proteins, these combined techniques allow forces to be measured that anchor the protein in the native membrane, as well as forces required to unfold the tertiary and secondary structure of the protein [1516], and the protein to be imaged at subnanometer resolution.