J. Mol. BioE. (1991) 221, 361-365


Structure of Immunoglobulin G by Scanning Tunnelling Microscopy Robin J. Leatherbarrow Department

of Chemistry, Imperial College of Science, Technology South Kensington, London SW?’ ZAY, U.K.

Margaret Division


of Mechanical and Optical Metrology, Teddington, Middlesex TWll

and Timothy SmithKline


National Physical OLW, U.K.


N. C. Wells?

Beecham Pharmaceuticals, The Frythe, Hertfordshire AL6 9AR, U.K. 14 September

and Medicine

1990; accepted 24 May



Scanning tunnelling microscopy (STM) has been used to examine the shape of individual immunoglobulin G (IgG) molecules deposited onto a graphite surface. IgG was chosen for this study as it has a well-characterized and distinctive three-dimensional structure. The micrographs clearly reveal the IgG molecule as trilobed, corresponding with the known structural organization of IgG. Comparison of these images with the structure of IgG determined by X-ray crystallography shows that the STM images are consistent with the crystal structure. This illustrates that STM is a valuable technique for examining protein structure, allowing rapid determination of the overall molecular shape that is consistent with more established techniques. Keywords: immunoglobulin

G; scanning tunnelling microscopy;

The immune response is mediated by antibodies, which act to bind a diverse range of foreign molecules. In turn, complexed antibodies are recognized by further components of the complement pathway or by cellular receptors. The predominant antibody is IgG (M, 150,000). This molecule has three discrete regions; antigen binding is via one or both of two identical Fab arms, whereas the effector functions are in the Fc region (Marquart & Deisenhofer, 1982). A flexible hinge links these two regions. Electron microscopy studies of negatively stained samples show IgG to be a T or Y-shaped molecule (Valentine & Green, 1967). X-ray crystallography provides atomic resolution for proteins, and for IgG the following structural data are available: crystals of IgG Kol reveal a Y-orientation for the Fab arms, but no electron density for the Fc region implying

flexibility of this region (Marquart et al., 1980). The IgG protein Dob was found to have a T-shape (Silverton et al., 1977), but Dob is a mutant protein wit,h a 15-residue deletion in the hinge region that is thought to restrict flexibility of the Fc relative to the Fab. Although Dob contains this deletion, the structure of the Fab and Fc portions is the same as for normal IgG. In addition, high resolution X-ray are available for individual Fab structures (Marquart et al., 1980) and Fc (Deisenhofer 1981) fragments obtained by proteolytic cleavage. Scanning tunnelling microscopy (STMt) (Binnig et al., 1982) allows visualization of molecular surfaces with unparalleled detail, and can give atomic resolution with suitable samples (Hansma et al., 1988). In biological systems, the organization of $ Abbreviations used: STM, scanning tunnelling microscopy; HOPG, highly-orientated pyrolytic graphite.

t Present address: Glaxo IMB SA. 14 Chodes Aulx, 1228. Plan-les-Ouates,








1991 Academic PreessLimited

R. J. Leatherbarrow



et al.

1. STM image showing several IgG molecules

macromolecules is of vital importance to function. STM has been used to image biological macromolecules including proteins and DNA (Hansma et al., 1988; Beebe et al., 1989; Cricenti et al., 1989; Dunlap & Bustamante. 1989; Edstrom et al., 1989; Welland et al., 1989; Driscoll et aZ., 1990). STM allows direct observation of biological samples without the need for shadowing with heavy metals. Although the theoretical basis for imaging nonconductive biological materials is not well understood, resolution of the order of 1 nm or less can be obtained for suitable samples (Cricenti et aZ., 1989; Dunlap & Bustamante, 1989; Driscoll et al., 1990). In order to test the applicability of STM to proteins, we used the structurally well-characterized IgG protein. IgG has a characteristic three-dimensional structure, and so allows us to evaluate the utility of STM on protein samples. The STM experiments used native samples of human IgG (Nova Biochemicals) air-dried onto the surface of freshlycleaved HOPG substrate (Le Carbone). The IgG used contained a mixture of the four different subclasses, but was predominantly IgGl. A total of 1 ~1 of protein solution (0,5mg/ml in 50mM-

on HOPG (200nm x 200nm)

Tris.HCl (pH 8)) was used for the experiment. It was important to use low concentrations of protein to minimize aggregate formation. STM images were recorded with a Nanoscope II (Digital Instruments, Inc.) in air using a platinum/iridium tip. The tunnelling current was 0.5 nA, the bias 20 mV. Tunnelling was initiated immediately after the liquid had evaporated. This ensured that any damage caused by the preparation or harsh conditions was minimized. We observed that during imaging the IgG molecules occasionally moved across the field of view during scanning; this problem is widely encountered in imaging bio-molecules on HOPG by STM (Salmeron et al., 1990). Molecules of IgG on a highly-orientated pyrolytic graphite (HOPG) surface were located by STM using a large scan area. Figure 1 shows a region of the surface that contains several IgG molecules. All the molecules were of the same size and had a characteristic trilobed shape. Figure 2 shows two representative IgG molecules at increased magnification. Three distinct lobes are clearly visible in all the images recorded. In view of the known structural organization of IgG, we conclude that the three lobes corre-




Figure (dark=0

2. STM image of 2 individual nm, light =4 nm).



spond to the two Fab arms and the Fc region. We found remarkable consistency between the shape of the individual molecules imaged by STM. The structure of the hinge-deleted IgG protein Dob, as determined by X-ray crystallography

(45 nm x 45nm).


is coded





(Silverton et al., 1977), is shown in Figure 3. The image of native IgG obtained from STM is remarkably similar to the data from X-ray crystallography. The degree of similarity gives us confidence that the STM technique is accurately reflecting the


R. J. Leatherbarrow

et al.

Figure 3. The structure of the hinge-deleted IgG Dob (Silverton et aE. 1977). The molecule is shown as a space-filled image, using a single sphere for each amino acid residue or hexose unit.

native conformation of the protein. It further indicates that the use of a graphite surface, which is a very convenient substrate for STM, is suitable for studying protein samples. The size of the IgG molecules (Fig. 2) is in reasonable, but not precise, agreement with the dimensions found from X-ray crystallographic studies. The length and width of the Fab arms, excluding solvent, in the X-ray structure are approximately 8.5 nm x 6 nm; in our images, the apparent dimensions are approximately 17.5 nm x 11.5 nm. This increase in size could be due to the presence of remaining solvent around the molecule, deformation by the tunnelling tip during imaging, imperfect tip geometry or an adsorption effect to the graphite surface. Information in the z axis is limited, due to the complex nature of the tunnelling effect with large molecules, but the approximate height of 3 nm is consistent with the X-ray data. Most importantly, the overall shape of the protein is very well reproduced in the STM experiment. Although other proteins have been studied by STM (Hansma et al., 1988; Edstrom et al., 1989; Welland et al., 1989), IgG provides an excellent system to test the quality of shape information derived from STM of proteins, as it has a very characteristic quaternary structure. The strong correspondence between the images obtained and the known structure of IgG suggests that this technique will be of great value in studying other protein systems. STM experiments need submicrogram quantities of protein, and high resolution images can be recorded in less than five minutes. The images are reproducible, and give

information consistent with the more established techniques used for studying protein structure. We thank Professor D. M. Blow, Dr J. M. Squire, Dr P. Luther and Professor A. Franks for helpful discussions, and Dr D. R. Davies for providing the co-ordinates of the IgG protein Dob.

References Beebe, T. P., Jr, Wilson, T. E., Ogletree, D. F., Katz, J. E., Balhorn, R., Salmeron, M. B. & Siekhaus, W. J. (1989). Direct observation of native DNA structures with the scanning tunneling microscope. Science, 243, 370-372. Binnig, G., Rohrer, H., Gerber, Ch. & Weibel, E. (1982). Surface studies by scanning tunneling microscopy. Phys. Rev. Letters, 49, 57-61. Cricenti, A., Selci, S., Felici, A. C., Generosi, R., Gori, E., Djaczenko, W. & Chiarotti, G. (1989). Molecular structure of DNA by scanning tunneling microscopy. Science, 245, 12261227. Deisenhofer, J. (1981). Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 29 and 28A resolution Biochemistry, 20, 2361-2370. Driscoll, R. J., Youngquist, M. G. & Baldwschwieler, J. D. (1990). Atomic-scale imaging of DNA using scanning tunneling microscopy. Nature (London), 346, 294-296. Dunlap, D. D. & Bustamante, C. (1989). Images of singlestranded nucleic acids by scanning tunnelling microscopy. Nature (London), 342, 204-206.

Communications Edstrom, R. D., Meinke, M. H., Yang, X., Yang, R. & Evans, D. F. (1989). Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy. Biochemistry, 28, 49394942. Hansma, P. K., Elings, V. B., Marti, 0. & Bracker, C. E. (1988). Scanning tunneling microscopy and atomic application to biology and force microscopy: technology. Science, 242, 209-216. Marquart, M. & Deisenhofer, J. (1982). The threedimensional structure of antibodies. Immunol. Today, 3, 160-166. Marquart, M., Deisenhofer, J. & Huber, R. (1980). Crystallographic refinement and atomic models of the intact immunoglobulin molecule Kol and its antigenbinding fragment at 3.0A and 1.9A resolution. J. Mol. Biol. 141, 369-391.


Salmeron, M., Beebe, T., Odriozola, J., Wilson, T., Ogletree, D. F. & Siekhaus, W. (1990). Imaging of biomolecules with the scanning tunneling microscope: problems and prospects. J. Vat. Sci. Technol. Sect. A, 8, 635-641. Silverton, E. W., Navia, M. A. & Davies, D. R. (1977). Three-dimensional structure of an intact human 74, immunoglobulin. Proc. Nat. Acad. Sci., U.S.A. 5140-5144. Valentine, R. C. & Green, P;. M. (1967). Electron microscopy of an antibody-hapten complex. J. Mol. Biol. 27, 615617. Welland, M. E., Miles, M. J., Lambert, N., Morris, V. J., Coombs, J. H. & Pethica, J. B. (1989). Structure of the globular protein vicilin revealed by scanning tunnelling microscopy. Int. J. Biol. Moxromnl. 11, 29-32.

Edited by A. Klug

Structure of immunoglobulin G by scanning tunnelling microscopy.

Scanning tunnelling microscopy (STM) has been used to examine the shape of individual immunoglobulin G (IgG) molecules deposited onto a graphite surfa...
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