International Journal of Advances in Science and Technology, Vol. 3, No.

1, 2012

Atomic Force Microscopic study of Myosin molecule on different substrates

Sunita Kumari
Assistant Professor, Department of Biotechnology, Post Graduate Government College for Girls, Sector 42, Chandigarh, drsunita31@gmail.com

Abstract
The present study reports the covalent and non covalent immobilization of myosin molecule on different substrates and their visualization using atomic force microscope. Myosin was immobilized on poly-Llysine, gold, mica and silicon-coated glass slides and their roughness was calculated from the images taken from AFM. AFM is a powerful tool for imaging biomolecules on a substrate. The myosin motor can be used for controlled transportation of cargo for nanotechnological applications, mimicking transportation processes in the living cell. Keywords: Myosin, atomic force microscope (AFM), immobilization, poly-L-lysine, gold, mica, silicon, biomolecular electronics and nanotechnology

1. Introduction
The last decade has seen an erupting development in the research of single molecules. The driving force behind single molecule research has been several fold: instrumental advancements in detector and force measuring technologies, the desire to understand the mechanism of action of biological machines and to manufacture similar machines of and nanometer dimensions, just to name a few. The AFM was originally designed to be an imaging tool (Binning et al, 1986). Modified from the design of STM, the AFM acquires topographic images by methodically scanning the specimen with a flexible cantilever that bends according to the contour of the surface. Atomic level resolution is acquired by translating the deflection of cantilever into and image map of surface height differences. Since mapping of the surfaces can be conducted in both air and aqueous environments, imaging studies of biomolecules are possible under near physiological conditions, thus, enabling researches the subtle details of biological structures such as biomembranes (Zasadzinski et al, 1988), bacteriorhodopsin (Butt et al, 1990) and DNA (Hansma et al, 1990). For imaging biomolecules by AFM, a proper immobilization onto a flat surface has to be concepted. For that, in general, four different methods are possible: 1) drying of the sample solution on a surface 2) physical binding of the molecule e.g. via counter ions or hydrophobic interaction to a surface (Thundat et al, 1992, Hansma et al, 1993) 3) chemical binding or cross linking of the molecule to a surface (Karrasch et al, 1993, Wagner et al, 1996) or 4) embedding of the molecular species in a 2-D array (Schabet et al, 1995). Two different approaches have been used based on the nature of the attachment made between sample and surface which can be covalent or non-covalent.

1.2 Covalent immobilization

In many cases single molecule and filamentous structure requires where a stable covalent bond is formed between chemical groups of the specimen and functionalities that are exposed at the substrate surface.

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International Journal of Advances in Science and Technology, Vol. 3, No.1, 2012 Covalent immobilization is an important strategy for those applications where displacement or desorption is a critical issue, but also when conditions for adsorption and biological activity are incompatible, or when the molecular on objects have to be integrated in complex supramolecular assemblies that include assembly processes and require well defined coupling steps. With respect to AFM applications, 3 aspects are critical for covalent immobilization strategies: i) the immobilization chemistry must be reproducible ii) the integrity and activity of the biomolecule should be affected by the chemical reaction with the substrate and iii) the surface must be atomically flat, hydrophilic, preferentially monofunctional, and should be negatively interfering with the scanning probe.

1.3 Myosin structure

The molecule of muscle myosin is a hexamer comprising two heavy chains (molecular weight ~200 kD) and four light chains (molecular weight ~20 kD). The N-terminal parts of the heavy chains form two globular heads, with two light chains associated with each head. Each myosin head contains the ATPase site and the actin-binding sites. The muscle myosin molecule is 150nm long and 10nm wide. In our present study, we immobilized myosin molecule on different substrates like lysine coated glass surface, gold, mica and silicon surfaces and imaged them with the help of atomic force microscope.

2. Materials and Methods

Rabbit skeletal myosin, Poly L- lysine coated glass slides and other biochemicals were procured from Sigma Aldrich and EDC (1-ethyl-3(3- dimethylaminopropyl)-carbodiimide) was procured from Pierce, USA.. Gold coated glass slides were procured from Thin Labs, CSIO, Chandigarh, India. In all the experiment microbes and pyrogen free deionized water having resistivity of 18.2 MegaOhm obtained from PureLab, Elga, UK, water purification system was used.

2.1 Immobilization of myosin on Poly-L-lysine coated glass surface

Myosin was immobilized on Poly-L-lysine coated glass. Poly-L-lysine coated glass slides (1cm x1cm) were washed with 500l of conjugation buffer (0.1M MES (2-[N-morpholino] ethane sulfonic acid), pH 4.5-5), then EDC (10mg/ml) was dispersed on lysine coated glass wafers and incubated for 15 minutes. Myosin was immobilized using the following procedure. 100l of EDC (10 mg/ml) was poured on the glass slide, after 15 minutes 200l of myosin (1mg/ml) was added into the reaction mixture on the glass slide and then incubated at room temperature for 2hr. After 2hr the slide was washed with MES buffer and then air dried. Then immobilization was confirmed using Atomic Force Microscope (Nanoscope II).

2.2 Dicing of solid substrates

The dicing of solid substrates (gold coated glass slide, glass slide, silicon surface) into squares (area: 1cmx 1cm) was carried out using Microautomation, USA Model No. 1100 Series at S.C.L., Mohali, India. Dicing of gold-coated glass slide and silicon slide were done from the side that did not have gold and silicon coating respectively.

2.3 Immobilization of myosin on gold-coated glass slide

Surface was activated by a quick 1-minute dip in Piranha solution (H2SO4 70%: H2O2 30 % in 3:1 v/v), followed by rinsing with distilled water for about 10 minutes. Again rinsed with ethanol and then dipped in 1.2 N NaOH for about 30 minutes and then washed with distilled water. Soaked in 1.2 N HCl for 15 minutes. Soaked in 50l of 12N HCl for 2 minutes. Then washed with distilled water followed by ethanol. Dried at 700C for about 30 minutes. Dipped in protein A (0.5 mg/ml) solution. Incubated overnight at room temperature. Next day the gold substrate was washed with phosphate buffer. Added 100l of 10mg/ml

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International Journal of Advances in Science and Technology, Vol. 3, No.1, 2012 freshly prepared EDC and left for 2 Hrs. at room temperature. Washed with conjugation buffer and added 50l of 2mg/200l myosin at 370C. Kept at room temperature for 2 hrs. Washed again with conjugation buffer (0.1M MES, pH 4.5-5.0).

2.4 Immobilization of myosin on mica surface

Following protocol was used for immobilizing myosin on mica surface: 100l of 0.05mg/ml myosin in HEPES buffer (50mM HEPES, 100mM KCl, 5mM MgCl2, 1mM DTT, pH 7.2) was deposited on a freshly cleaved mica surface, and molecules were left to adsorb on the surface for 1 hr. at room temperature. The mica surface then rinsed with HEPES buffer. Then the surface was fixed with 0.1% gluteraldehyde for 3 minutes and rinsed again with HEPES buffer. Finally, the wafer was dried in dry nitrogen flow for AFM imaging.

2.5 Immobilization of silicon surface myosin on

The silicon wafers were cleaned with a mixture of 30% hydrogen peroxide (H2O2) and concentrated sulfuric acid (H2SO4) (1: 3v/v) for 30 min. After thoroughly rinsing with water and pure ethanol, the slides were stored in pure ethanol before use.

2.6 Silanization of cleaned silicon wafer surfaces with APTES

The cleaned wafers were reacted with a fresh ethanol solution of APTES (5% APTES, 5% water and 90% pure ethanol) for 1h at room temperature, followed by the rinsing with water three times and pure ethanol three times, and then stored in pure ethanol.

2.7 Covalent immobilization of myosin

The silicon surfaces with APTES were reacted with a 2.5% solution of gluteraldehyde in PBS buffer for 2h, followed by rinsing with PBS buffer. The gluteraldehyde surfaces were then placed into 0.1mg/ml myosin solution with the addition of 1% Tween 20 at room temperature. The surfaces were washed with PBS buffer and the remaining aldehyde groups on the surfaces were deactivated with 1M ethanolamine for 30min

2.8 Biomolecular imaging by Atomic force microscopy

The structural morphology of immobilized myosin was studied using contact and non-contact mode AFM, Nanoscope II, Digital Instruments Inc., USA. Imaging was done in air using 0.7-micron AFM head. The sample was placed on xyz-piezo-translator and scanned by a sharp diamond tip mounted on a gold-coated 200m triangular silicon nitride microfabricated cantilever (force constant = 0.6 N/m). The force between the tip and the sample was usually in the range of 10-9 N. Images consist of 400 scans of 400 pixels each. Typical image acquition time was 150-200 s/scan depending upon scan speed. All samples were scanned in contact force mode using non-contact mode AFM.

3. Results and discussion

The immobilization of functional proteins on flat surfaces is of crucial importance for studying their interaction with ligands and examining their structure by means of electron and scanning probe microscopy and other biophysical techniques requiring a solid interface. We have used heterobifunctional cross linker EDC to immobilize myosin on poly-L-lysine coated glass slide. The surface characterization of myosin on lysine coated glass slide was done by taking AFM images in contact mode in air and EDC to immobilize myosin on poly-L-lysine coated glass slide. The surface characterization of myosin on lysine coated glass slide was done by taking AFM images [Figure 1] in contact mode in air calculated the roughness of the surface.

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International Journal of Advances in Science and Technology, Vol. 3, No.1, 2012

1 (a) Glass slide coated with lysine (roughness 0.5nm)

1 (b) EDC coated on lysine coated glass slide (roughness 1.2-1.4nm)

(c)

1 (c) Myosin molecules immobilized with EDC (roughness 2.4-4.5nm).

Figure 1. Contact mode AFM images of myosin cross linked to lysine coated glass slide The AFM image of gold-coated glass slide taken in non contact mode (figure 2) was homogeneously flat with significantly no heights on the surface; the slide was uniformly coated with protein A. The spatial distribution of protein a on the gold surface was limited by several factors like steric repulsion between protein A molecules.

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2 (a) (a) protein A on gold coated glass slide ( roughness 0.10.2nm)

2 (b) b) EDC crosslinked to protein A ( roughness 1.4-2.5nm)

2 (c) c) myosin cross linked to EDC ((roughness 2.6-4.5nm)

Figure 2: Non-contact AFM images of

With mica, an active surface is very conveniently obtained by cleaving the layered mica crystals prior to specimen adsorption. Figure 3(a) shows the non-contact AFM image of mica surface. There are no regular features on it. The topography image in figure 3(b) shows the myosin immobilized on mica surface and imaged in air. On this area of 9.5m and 3m myosin heads can be resolved.

(a) mica surface

(b) myosin immobilized on mica surface Figure 3. AFM images

The myosin filaments are arranged in a regular symmetry and the myosin heads are protruding from the surface. All the heads are protruding in a single direction and in a similar orientation. This orientation is a requisite to use mica surface for in vitro assays. Surface chemical modification is the first step of the process leading to immobilization of biomolecules on surface. Although it is possible to chemically modify different types of surfaces, silicon surface do not interfere with binding chemistries. Silicon surfaces also present a very low root mean square (RMS) roughness, which is essential for single molecule studies. AFM images were taken and the RMS roughness

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International Journal of Advances in Science and Technology, Vol. 3, No.1, 2012 of the silanized surfaces was calculated to be 0.19nm. The topography image in figure 4 shows the surface of silicon coated with myosin and the area scanned is 1.4m.

Figure 4. AFM image of myosin immobilized on silicon surface Comparing the measured dimensions with the dimensions of the known structure, we assume that these are myosin heads which are at a height of 120nm from the surface. The length of myosin filament is estimated to be 150nm and the heads are 20nm in diameter.

4. Conclusion
These surfaces can be integrated with biological part in future for hybrid nanodevice applications.The ability of AFM to image, address and probe single molecules opens fascinating possibilities in order to observe, handle and manipulate individual molecules. As instrumentation for AFM improves and a wider range of equipment becomes commercially available, it is likely that cell biologists will routinely use AFM to measure molecular interaction forces. Actin-myosin acts as a linear motor and has various applications in the nanomechanical devices and drug delivery. In vitro it can be used for transportation of cargos in a factory on a chip application and nanofabricated structures to power the nanomechanical devices. Anchoring the biological components is necessary to provide the leverage necessary for movement. For practical realization of simple, costeffective and easy to fabricate drug delivery and nano-switches, the controlled motility of these motor proteins on semiconductor surfaces is to be conducted.

5. References
1.Binnig G, Quate C F, Gerber C. Atomic force microscope. Phys Rev Lett, (1986), 56(9): 930-933 2.Zasadzinski J.A., Schneir J., Gurley J., Elings V., and Hansma P.K. (1988). Scanning tunneling microscopy of freeze-fracture replicas of biomembranes. Science 239: 1013-1015 3.Butt H.J., Downing K.H., and Hansma P.K.(1990). Imaging the membrane protein bacteriorhodopsin with atomic force microscope. Biophys. J. 58: 1473-1480

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International Journal of Advances in Science and Technology, Vol. 3, No.1, 2012 4.Hansma H.G., Vasenka J., Siegerist C., Kelderman G., Morret H., Sinsheimer R.L., Elings V., Bustamante C and Hansma P.K. (1992). Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science 256: 1180-1184 5.Thundat, T., Allison, D.P., Warmack, R.J., Brown, G.M., Jacobson, K.B., Schrick, J.J., and Ferrell, T.L. (1992). Atomic force microscopy of DNA on mica and chemically modified mica. Scanning microscopy 6, 911-918. 6.Hansma, H.g., Sincheimer, R.L., Grappe, J., Bruice, T.C., Elings, V., Gurley, G., Bezanilla, M., Mastragelo. I.A., Hough, P.V.C., and Hansma, P.K..(1993). Recent advances in atomic force microscopy of DNA. Scanning 15, 296-299. 7.Karrasch, S., Dolder, M., Schbert, F., Ramsden, J., and Engel, A. (1993). Covalent immobilization of native biomolecules onto Au(III) via N-hydrosuccinimide ester functionalized self assembled monolayers for scanning probe microscope, Biophys. J.70, 2052-2066. 8.Hansma, H.G, Hoh, J.H. (1994) Biomolecular microscope. Annu Rev Biophys Biomol Struct. 23:115139 imaging with the atomic force

9.Schabert, F.A., Henn, Ch., and Engel, A.(1995). Native Escherichia coli OmpF porin surfaces probed by atomic force microscope, Science 268, 92-94. 10. Bustamante, C. and D. Keller (1995). Scanning force microscopy in biology. Physics Today (Dec.): 3238 11.Engel, A., Schoenenberger, C-A., and Mller, D.J. (1997). High resolution imaging of native biological sample surfaces using scanning probe microscopy. Curr. Opin. Struct. Biol. 7; 279-284. 12. Ellis Bagga, Sunita Kumari, Rajesh Madan, Rakesh Kumar, R P Bajpai, Lalit M Bharadwaj. Covalent immobilization of Myosin for in vitro motility of actin. Pramana-journal of physics. 65, No. 5, (2005) 967-972. 13.Kumar, R., Shukla, A.K., Bagga, E., Kumari, S., Bajpai, R.P., and Bharadwaj, L.M. (2005) 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide interference with Lowry method. Anal. Biochem., 336, 132-134.

Authors Profile

Dr. Sunita Kumari did her Masters in Biotechnology from Jawahar Lal Nehru University. She received her Ph.D degree from Panjab University, India in 2007. She is currently working as an Assistant Professor in Department of Biotechnology, Post Graduate Govt. College for Girls, Chandigarh. She is having teaching experience of 6 years. Her major research Interests is nanotechnology.

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