Scanning tunnelling microscopy of Z-DNA

100

Scanning tunneling microscopy has been used to demonstrate that a spiral structure based on -reverse turns is adopted by the repeat sequences present in a group of wheat gluten proteins. This structure is smilar to the «spiral formed by a synthetic polypentapeptide based on a repeat sequence present in elastin. Wheat gluten and elastin are both elastomeric and it is possible that the spiral structure contributes to this property.Scanning tunneling microscopy (STM) and the derivative scanning probe techniques can produce high-resolution images of structures at the atomic and molecular levels. Already used as a tool in surface science (1), many of the properties of STM have great potential for the study of biopolymers. The STM can operate in air and even in liquid to image uncoated and unstained biomolecules deposited on a conducting surface. This allows biopolymers in their native hydrated state to be imaged (2-5). STM images of DNA have confirmed the details of the helical structure established by x-ray diffraction, giving confidence in this form of microscopy (6-10). STM can, therefore, be used to image structures that, to our knowledge, have not been described by other techniques. In the present study STM has been used to study the structure of a high molecular weight (HMW) subunit protein from wheat gluten for which an unusual structure has been predicted from the amino acid sequence and on the basis of other physicochemical studies.The HMW subunits of wheat gluten appear to be largely responsible for the elastic behavior of dough. Analyses of genomic clones encoding several subunits have shown that they have similar structures (11,12). Each protein consists of a central repetitive domain, varying in length from about 640 to 830 residues, flanked by shorter nonrepetitive N (81-104 residues)-and C (42 residues)-terminal domains. The HMW subunits are classified into two groups on the basis of their molecular weights, x-types (molecular weights in the range 83,000-88,000) and y-types (molecular weights in the range 67,000-74,000). The central repetitive domains are based on three motifs. Hexapeptides (consensus Pro-Gly-Gln-GlyGln-Gln) and nonapeptides (consensus Gly-Tyr-Tyr-Pro-ThrSer-Pro/Leu-Gln-Gln) are present in both x-and y-type subunits and tripeptides (consensus Gly-Gln-Gln) in x-type subunits only. The central domains are predicted to form regularly repeated (-reverse turns (13). Further evidence for the presence of (-reverse turns has come from spectroscopic (circular dichroism and Fourier-transform infrared) studies of synthetic peptides corresponding to the repeat motifs present in both x-and y-type subunits (14). Hydrodynamic studies of a single purified x-type HMW subunit from durum wheat showed an extended rod-like conformation and it was proposed that the (-reverse turns form a loose spiral (15). Modeling of several proteins containing (-reverse turns has indicated the possibility of spiral structures (16), and detailed studies of a synthetic polypentapeptide based on the repeat motif of e...

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confidence: 83%

Scanning tunneling microscopy of a wheat seed storage protein reveals details of an unusual supersecondary structure.

Miles

Carr

McMaster

et al. 1991

100

“…Several recent studies have examined the possibility of imaging DNA by scanning tunneling microscopy (STM) (1)(2)(3). However, the ultimate goal-to read the code contained in the strands-has not yet been achieved.…”

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confidence: 99%

Two-dimensional ordering of the DNA base guanine observed by scanning tunneling microscopy.

Heckl

Smith

Binnig

et al. 1991

142

Guanine, one ofthe four DNA bases, has been observed by tunneling microscopy to form a two-dimensional ordered structure on two crystalline substrates, graphite and MoS2. The two-dimensional lattice formed by guanine is nearly identical on the two surfaces, and heteroepitaxy appears to be the growth mechanism in both cases. Although the resolution of molecular details is superior for the graphite substrate, the simpler results on MoS2 are not only easier to interpret but also facilitate the understanding of the more complex images on graphite. We propose that the interfacial structure is composed of linear chains of hydrogen-bonded molecules aligned into a closely packed two-dimensional array.Several recent studies have examined the possibility of imaging DNA by scanning tunneling microscopy (STM) (1-3). However, the ultimate goal-to read the code contained in the strands-has not yet been achieved. To do so it must be possible to recognize clearly and distinguish between the four bases of the genetic code. One essential requirement for imaging small organic molecules by STM is to find experimental preparation conditions whereby the molecules stick firmly to the substrate and form a highly stable layer. This is necessary to withstand the forces of the STM tip during imaging. Compared to the binding of complete DNA strands to the basal planes of MoS2 or graphite, the adsorption of "naked" nucleotide bases is favored by their greater hydrophobicity and, as is shown in this report, by their ability to register with the substrate, thus forming a stable twodimensional ordered array. No signs of mobility were observed during investigations, which lasted up to 1 h in a single region. That such an array can form is probably due to the intermolecular hydrogen bonding capability of the DNA bases (4).Ordered molecular layers of organic molecules, such as benzene (5), alkanes (6), or liquid crystals (7,8), are the most prominent examples to have been imaged recently by STM at atomic resolution. STM results of adenine, one of the four DNA bases, have recently been obtained on graphite by Allen et al. (9). Using a sample preparation technique similar to theirs (9), we prepared samples of guanine on the surfaces of natural MoS2 crystals and highly oriented pyrolytic graphite. We often observed steps from the bare substrate to monolayers of guanine. The results presented in this paper were obtained on such monolayer islands. The character of the steplines provides additional information since the orientation of the molecular lattice with respect to the substrate can be measured there. In the STM images the bases look quite different depending on whether they are deposited on MoS2 (Fig. 1) or on graphite (see Fig. 3). The main differences are that on MoS2 the bases appear as distinct well-isolated nearly structureless blobs; on graphite more details become visible, although it is still not possible to determine the 8003The publication costs of this article were defrayed in part by page charge payment. This article must ...

“…Fragments ofnatural and synthetic DNA have been imaged by STM on highly oriented pyrolytic graphite (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) and gold (12,13) surfaces. However, because topographic contrast is generally used to locate these extremely fine (2-nm diameters) molecules by scanning probe techniques, the DNA mounting substrate must be extremely flat.…”

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confidence: 99%

Immobilization of DNA for scanning probe microscopy.

Allison

Bottomley

Thundat

et al. 1992

Reproducible scanning e microscope and atomic force microscope images of entire molecules of uncoated pasmid DNA chemically bound to surfaces are presented. The chemicaily mediated immobilization of DNA to surfaces and subsequent snning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with nning probe microscopes, can be prevented. (12,13) surfaces. However, because topographic contrast is generally used to locate these extremely fine (2-nm diameters) molecules by scanning probe techniques, the DNA mounting substrate must be extremely flat. Surfaces with atomic flatness are ideal but usually lack sites that might anchor DNA either mechanically or chemically, thereby preventing displacement or removal by the scanning probe tip. Attempts to immobilize DNA on surfaces using electrochemical methods (14, 15) and covalent linking (13, 16, 17) of DNA to surfaces have been successful, but to date only images of DNA fragments have been reported. Evidence for improved surface binding came from examination ofa surface frequently imaged with the AFM. Mica, although atomically flat, is an insulator and therefore cannot be imaged with the STM but is well suited for the AFM. By simply drying a drop of a DNA-containing buffer solution onto freshly cleaved mica, one can routinely obtain AFM images of DNA strands within minutes (18-21), perhaps stabilized by hydrogen bonding of Si-OH to DNA. Recently a technique that replaced potassium ions in the mica surface with magnesium ions, promoting stronger binding of DNA to mica resulting in improved AFM images, has been described (22)(23)(24). Since the forces between the probe and the surface are roughly comparable in STM and AFM, the AFM work gave us hope that with a properly modified conducting surface to stabilize DNA, routine imaging of DNA by STM should be possible. Using an approach that allows a thiol group at one end of an organic molecule to bind to a gold surface (25) and a charged head group at the other end to attract and immobilize the polyanionic DNA for STM imaging (26) we present the STM images of entire plasmid molecules. METHODSPlasmid DNA (3204 base pairs, pBS+ from Stratagene) was extracted from Escherichia coli, concentrated by cesium chloride/ethidium bromide equilibrium centrifugation, ethanol precipitated, and resuspended in 10 mM Tris.HCl/0. 1 mM EDTA (TE) at pH 7.5 to a final concentration of 500 gg/ml (27). Circular DNA was relaxed by x-ray (70,000 rads; 1 rad = 0.01 Gy) mediated single-strand nicking of supercoiled molecules. Linearized plasmid was obtained by treatment with the restriction endonuclease Sma I. Agarose gel (0.8-1% agarose) electrophoresis was used to differentiate supercoiled from relaxed plasmid and linearized DNA (27). Relaxed plasmid DNA was radiolabeled by nick-translation in the following reaction mixture: 35 pl of DNA (500 Ag/ml), 5 pl (10x) of NT buffer (500 mM Tris-HCl, pH 7.5/50 mM MgCl2/100 mM 2-mercaptoethanol/1 mg of nuclease-...