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Imaging Individual Chaperonin and Immunoglobulin G Molecules with Scanning Tunneling Microscopy S. L. Tang* and A. J. McGhie DuPont Central Science and Engineering Laboratory, Experimental Station, Wilmington, Delaware 19880-0356 Received July 31, 1995. In Final Form: November 22, 1995X We have obtained nanometer-scale scanning tunneling microscope images of individual, randomlyspaced immunoglobulin G (IgG) and chaperonin molecules in ultrahigh vacuum. The methodology developed for routine imaging includes the use of a sputter-deposited Au(111) substrate for deposition, tunneling tip with diameters smaller than 10 nm, very dilute concentration of protein molecules in a volatile buffer for molecular deposition, and a vacuum environment for preserving the molecules for imaging for about 2 weeks. The imaged structures of both molecules agree well with X-ray data. Compression of the vertical dimension of the molecule was severe only in the relatively thick (14.6 nm) chaperonin but not in the thin (∼4 nm) IgG. Effects such as degradation and cleavage of individual molecules were also observed. An evaluation of the existing proposed imaging mechanisms is presented.
Introduction The structures of individual protein molecules at nanometer-scale resolution have been traditionally studied with high-resolution electron microscopy. It is a valuable technique for gaining insight on the functional structures of proteins and their complexes because ∼99% of the known molecules do not readily form crystals for X-ray studies.1 Transmission electron microscopy (TEM), the most established electron microscopy technique for proteins studies, utilizes techniques such as negative staining,2 cryogenic sample preparation,3 etc. for imaging with a high-energy electron beam. The scanning tunneling microscope (STM) and the atomic force microscope (AFM) have the advantage of not requiring these cumbersome and potentially damaging experimental procedures, and yet allowing the viewing of these molecules at nanometer scale in real space. Efforts in imaging biological molecules have been directed primarily toward the AFM because of its convenient capability to image molecules under physiological conditions. Indeed the AFM has produced some spectacular images4 of randomly spaced protein molecules in buffer solution. However, it is by no means a fully developed technique generally applicable to all protein molecules. For example, efforts, including our own, to obtain AFM images of the widely studied immunoglobulin G (IgG) molecules have yet to overcome a major stumbling block: the large lateral force exerted by the tip precludes any meaningful structural interpretation of the images. Under specific experimental conditions detailed below, STM images of IgG and another protein molecule, chaperonin, were quite regularly obtained. These results allow us to discuss the strengths and weaknesses of STM imaging of protein molecules. * Author to whom all corresponce should be addressed. E-mail:
[email protected]. dupont.com. Tel: (302) 695-9056. Fax: (302) 695-1664. X Abstract published in Advance ACS Abstracts, February 1, 1996. (1) Chan, H. S.; Dill, K. Physics Today 1992, 45 (2), 24. (2) Schmidt, M.; Rutkat, K.; Rachel, R.; Pfeifer, G.; Jaenicke, R.; Viitanen, P.; Lorimer, G.; Buchner, J. Science 1994, 265, 656. (3) Chen, S. A.; Roseman, M.; Hunter, A. S.; Wood, S. P.; Burston, S. G.; Ranson, A. A.; Clarke, A. R.; Saibil, H. R. Nature 1994, 371, 261. (4) Yang, J.; Tamm, L. K.; Tillack, T. W.; Shao, Z. J. Mol. Biol. 1993, 229, 286.
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Sample Preparation and Experimental Procedures A. The Protein Molecules. The IgG molecules were from a monoclonal culture5 of mice which contained ∼70% IgG. IgG, a class of antibody molecules, has been extensively studied with X-ray crystallography and electron microscopy.6 It is made up of two heavy chains (∼50 kDa each) and two light chains (25 kDa each). These chains are arranged in a “T” or “Y” shape with two identical “Fab” arms and an “Fc” arm. The length and width of each arm from X-ray measurements7 are ∼8.5 and ∼6 nm, respectively. The height of the molecule from TEM measurements8 is ∼4 nm. A schematic drawing of the IgG molecule is shown in Figure 1a. The chaperonin molecules used in this study were isolated and purified from E. coli, the procedure for which is described in ref 9. Chaperonin is a cellular protein of topical interest because of its role in assisting the folding of polypeptides by preventing the aggregating and precipitating of partially folded and unassembled proteins.10 The structures of chaperonins and their complexes with other protein substrates have been intensely studied with TEM2,3,11-13 and X-ray crystallography14,15 in the past few years. From the most recent X-ray study,15 chaperonin, also known as GroEL, Hsp 60, or cpn 60, is found to be an oligomeric complex of 14 identical polypeptides (a 14-mer) of 547 residues each, with a molecular weight for the entire molecule of >800 kDa. The 14 subunits are arranged in two equal layers, each having seven subunits forming a ringlike structure. The ring is ∼13.7 nm in diameter, and the height of the two layers is ∼14.6 nm. A schematic view of the chaperonin molecule is shown in Figure 1b. The distinctive shapes of IgG and chaperonin (5) The IgG sample was obtained from Dr. K. Hardman of the DuPontMerck Pharmaceutical Company. The molecules were isolated using standard procedures as described in Voss, E. W. Fluoroscein Hepten: an Immuological Probe; CRC Press: Boca Raton, FL, 1987. (6) Burton, D. R. In Molecular Genetics of Immunoglobulin; Calabi, F., Neuberger, M. S., Eds.; Elsevier: Amsterdam, 1987; pp 1-50. (7) Wells, T. N. C.; Stedman, M.; Leatherbarrow, R. J. Ultramicroscopy 1992, 42, 44. (8) Valentine, R. C.; Green, N. M. J. Mol. Biol. 1967, 27, 615. (9) Viitanen, P. V.; Lorimer, G. H.; Seetharam, R.; Gupta, R. S.; Oppenheim, J.; Thomas, J. O.; Cowan, N. J. J. Biol. Chem. 1992, 267, 695. (10) Ellis, R. J.; Hemmingsen, S. M. Trends Biochem. Sci. 1989, 14, 339. (11) Hendrix, R. W. J. Mol. Biol. 1979, 129, 375. (12) Hohn, T.; Hohn, B.; Engel, A.; Wurtz, A.; Smith, P. R. J. Mol. Biol. 1979, 129, 359. (13) Ishii, N.; Tagushi, H.; Sumi, M.; Yoshida, M. FEBS Lett. 1992, 299, 169. (14) Svensson, L. A.; Surin, B. P.; Dixon, N. E.; Spangfort, M. D. J. Mol. Biol. 1994, 235, 47. (15) Braig, K.; Otwinowski, Z.; Hegde, R.; Boisvert, D. C.; Joachimiak, A.; Horwich, A. L.; Sigler, P. B. Nature 1994, 371, 578.
© 1996 American Chemical Society
Imaging Individual Molecules with STM
Langmuir, Vol. 12, No. 4, 1996 1089
Figure 2. 400 × 400 nm2 scan of the sputter-deposited Au(111) surface without molecular deposition. Atomically flat terraces marked by holes a few atoms deep were separated by monatomic and multiple steps. The gray scale bar is in nanometers. The tip bias voltage was 0.4 V, and the current was 0.2 nA.
Figure 1. (a) Schematic drawing of the IgG molecule showing the two Fab arms and the Fc arm. The dimensions of each arm are approximately 6 × 8.5 × 4 nm3. (b) Schematic drawing of the top view and the side view of the Chaperonin molecule. In the top view, a donut-shape ring with 7-fold symmetry is shown. The side view shows that the molecule is made up of two layers of equal thickness. A detailed description of the structure of the molecule can be obtained in ref 15. make them relatively straightforward to distinguish from background structures in STM images. By including two molecules with distinct shapes in this study, we provide an internal check for the capability of the STM to reproduce the characteristic shapes of protein molecules. B. Experimental Methods. Because of the many potential pitfalls present in imaging biological molecules with the STM, we describe our experimental conditions in great detail below to show how we avoided or minimized these pitfalls. The crucial components of our STM experiments are the following: (1) Sputter-Deposited Au(111) Film on Mica at 450 °C as Substrate for Deposition of the Molecules. The sputter-deposited Au(111) film thickness was ∼200 nm. The gold surface contains a large number of atomic-scale steps, troughs, and terraces. These monatomic steps and atomic-scale troughs serve two purposes. First they are readily identifiable such that molecular features can be easily distinguished from substrate structures. It was inevitable that occasionally some features on the surface did not resemble easily recognizable physical defects on the surface, but rather looked like mounds that might be attributable to protein materials. Typically these mounds did not change or move on the surface with repeated scanning, whereas what we identified as protein molecules and are reporting in this article did. We use the mobility and fragility of the features we imaged to help us further discriminate between substrate gold defects and surface molecular deposits. Second, they provide physical defects for anchoring the compact, globular protein molecules which otherwise have a tendency to be moved on a smooth surface. Figure 2 shows a 400 × 400 nm2 image of the uncovered Au(111) surface. Atomically flat terraces, monatomic and multiple steps, and pits that are roughly two atoms deep are the predominant features on this substrate. This physical trapping method works well in our STM investigations but does not hold molecules
strongly enough for AFM studies. Almost all of the molecules we imaged were found near or on top of physical defects on the substrate. (2) Very Sharp (
