Atomic force spectroscopy was used to study interaction strengths between bacterial antigens and receptors on macrophages. This method allowed for a direct comparison of the interaction strengths in different systems studied at the level of single molecules.
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.
One influential parameter which mediates interactions between many types of molecules and biological membranes stems from the lumped contributions of the transmembrane potential, dipole potential and the difference in the surface potentials on both sides of a membrane. With relevance to cell physiology, such electrical features of a biomembrane are prone to undergoing changes as a result of interactions with the aqueous surrounding. Among the most useful tools devoted to exploring changes of electrical parameters of a lipid membrane induced by certain extracellular ions, lipid composition, and embedded membrane peptides and proteins, are spectroscopic imaging and the inner field compensation (IFC) method. In this work we layout the principles of a fully computerized version of the IFC method, which makes it more readily available to users. As a direct application, we deployed this improved version of the IFC method to time-resolve changes induced by alamethicin monomers upon membrane dipole potential, following their aggregation within an artificial lipid membrane. Intriguingly, even prior crossing the membrane core, the membrane-bound alamethicin monomers are shown to significantly increase the dipole potential of the monolayer they reside in. Such data further emphasize the yet less-explored interplay between membrane-based protein and peptides, and the membrane dipole potential.
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