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1

Atomic Force Microscopy and Quartz Crystal Microbalance Study of the Lectin-Carbohydrate Interaction Kinetics

100%

Lebed K., Kulik A., Forró L., Lekka M.

Acta Physica Polonica A

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2007

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vol. 111

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issue 2

273-286

EN

Two analytical methods, atomic force microscopy and quartz crystal microbalance, were applied to the study of the reaction kinetics occurring between concanavalin A and carboxypeptidase Y, presenting the specific lectin-carbohydrate recognition. The dissociation rate constants for concanavalin A-carboxypeptidase Y complex obtained using both atomic force microscopy and quartz crystal microbalance were of the same order of magnitude: k_{diss}=0.170± 0.060 s^{-1} and k_{diss}=0.095±0.002 s^{-1}, respectively. In addition, each method alone aided in determining other parameters characterizing the studied interaction. Quartz crystal microbalance permitted us to estimate the association rate (k_{ass}=(5.6 ±0.1)×10^4 M^{-1} s^{-1}) and the equilibrium (K_a=(0.59×0.01)×10^6 M^{-1}) constants for the binding process occurring between concanavalin A and mannose residues of carboxypeptidase Y under given experimental conditions. Atomic force microscopy in force spectroscopy mode enabled the determination of the energy barrier position of r=2.29±0.04 Å characterizing the dissociation of concanavalin A- carboxypeptidase Y molecular complex. The presented results show that both atomic force microscopy and quartz crystal microbalance can be used to determine quantitative parameters characterizing the specific molecular interaction. Both methods can be easily combined for complementary and/or alternative studies of a chosen molecular interaction. By preparing the samples in the same manner the direct comparison between the data obtained via atomic force microscopy and quartz crystal microbalance can be made.

2

The Increase in Protein Contour Length Depends on Mechanical Unfolding Conditions

88%

Dąbrowska A., Lebed K., Lekka M., Kulik A., Forró L.

Acta Physica Polonica A

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2008

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vol. 113

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issue 2

753-762

EN

Many proteins in alive organisms have a domain structure providing them the possibility to reversible unfolding, which seems to play an essential role in those processes occurring in tissues which are controlled by mechanical cellular tension. In this work the atomic force microscopy was applied to investigate the mechanical properties of the single molecules of fibronectin, a protein participating in the important mechanical processes in extracellular matrix. The results showed that the conditions of mechanical stretching influence not only the force required to unfolding of a domain but also the increase in protein contour length induced by such unfolding event. Two mean values of the increase in length (called shortly the unfolding length) L_1 and L_2, were obtained and ascribed to unfolding of either the whole fibronectin domain of type III (L_2) or its fragment (L_1). Both unfolding lengths revealed similar dependence on the stretching conditions. This experimental observation of increase in unfolding length with increasing loading rate was successfully described with a combination of two theoretical models (Bell model and the worm-like-chain model), previously used separately in the analysis of protein unfolding. The general mechanical property of fibronectin domains was emphasized and proposed as a potential determinant of the cellular adhesion.

3

Study of Adhesion Interaction Using Atomic Force Microscopy

76%

Gryboś J., Pyka-Fościak G., Lebed K., Lekka M., Stachura Z., Styczeń J.

Acta Physica Polonica A

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2004

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vol. 105

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issue 5

501-510

EN

An atomic force microscope is a useful tool to study the interaction forces at molecular level. In particular the atomic force microscope can measure an unbinding force needed to separate the two single molecule complexes. Recent studies have shown that such unbinding force depends linearly on the logarithm of the applied loading rate, defined as a product of scanning velocity and the spring constant characterizing the investigated system (cantilever vs. surface). This dependence can be used to study the energy landscape shape of a molecular complex by the estimation of energy barrier locations and the related dissociation rates. In the present work the complex consisting of ethylene(di)aminetetraacetic acid and the bovine serum albumin was measured. The dependence between the unbinding force and the logarithm of the loading rate was linear. Using the Bell model describing the dissociation of the above molecules caused by the action of the external bond breaking force, two parameters were estimated: the dissociation rate and the position of the energy barrier needed to overcome during a transition from a bound to unbound state. The obtained results are similar to those obtained for a typical ligand-receptor interaction.

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3 Acta Physica Polonica A

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3 Lebed K.

3 Lekka M.

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1 Gryboś J.

1 Pyka-Fościak G.

1 Stachura Z.

1 Styczeń J.

Atomic Force Microscopy and Quartz Crystal Microbalance Study of the Lectin-Carbohydrate Interaction Kinetics

The Increase in Protein Contour Length Depends on Mechanical Unfolding Conditions

Study of Adhesion Interaction Using Atomic Force Microscopy