A I Archakov
Institute of Biomedical Chemistry, Russia
Title: AFM physicochemical properties and activity of single protein molecules of CYP 102A1 (BM3)
Biography
Biography: A I Archakov
Abstract
Atomic force microscopy (AFM) is a nanotechnological multifunctional molecular platform for measuring of physicochemical and functional properties of single proteins molecules. AFM was used for visualization of oligomeric state, activity, elasticity and electron transfer of single molecules of CYP 102A1 (BM3). It was shown that BM3 in water solution exists as monomer and different oligomers by use of sharp and super sharp AFM probes. Functional activity of single monomers and oligomers of BM3 was measured by AFM as well. The BM3 height fluctuations amplitude (HFA) during catalytic cycle is much larger than the HFA of the enzyme molecules in the resting state. It was found that an average HFA of dimers P450 BM3 during catalytic cycle increased up to 5.0±2Å·s-1 that was 2.5 times larger than a HFA of P450 BM3 in the resting state. It was obtained that the HFA of immobilized on mica cytochrome P450 BM3 depends on temperature, and 22ËšC is a peak of this temperature profile. Mass spectrometry (MS) measurements were used to obtain a time course of a hydroxylation product of lauric acid oxidation during the enzymatic reaction of P450 BM3 in two cases: when enzyme was solubilized in the volume and when it was immobilized on the mica chip. In both cases the number of enzyme molecules was ≈1010, and the kinetics was linear during the first 10 minutes. It was shown that in the case of solubilized enzyme kcat=10-3 s-1, and in the case of immobilized enzyme kcat=0.4·10-3 s-1 that was 2.5 times less than the first one. The purpose of our work was to find a relationship between enzyme HFA and its catalytic activity. Therefore, AFM data was analyzed together with MS data and the following equation was obtained: kcat= β· (exp(ΔA/ A0)-1, where kcat – is a catalytic rate constant (s-1); β is a proportionality factor (s-1); ΔA = Δ Ä€ – A0 (Å), where Ä€ and A0 are the average amplitudes of P450 BM3 height oscillations in the active and resting states, respectively. The value β = 5·10-6 s-1 was calculated from time dependence of reaction curves measured by AFM and MS. Elasticity of single protein was measured based on deformation of this protein under AFM probes with various radii of curvature. Young’s modulus of BM3 molecules depends on AFM modes. Based on the obtained data, the following conclusions may be made: the enzyme catalytic activity of single molecules can be measured as a HFA of BM3 oscillation during catalytic cycle.