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Nano Research & Applications
ISSN 2471-9838
Advanced Nano 2017
Notes:
Page 32
September 11-12, 2017 Amsterdam, Netherlands
20
th
International Conference on
Advanced Nanotechnology
AFM characterization of the physicochemical
properties and activity of single protein
molecules of CYP 102A1 (BM3)
Yuri Ivanov, A I Archakov
and
Bukharina N S
Institute of Biomedical Chemistry, Russia
A
tomic force microscopy (AFM) is a nano-technological
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, dimer, trimer,
tetramer and oligomers of higher order by use sharp and
super sharp AFM probes. Functional activity of single
monomers and oligomers of BM3 was measured by AFM.
The height BM3 fluctuations amplitude during catalytic
cycle is much larger than the height fluctuations amplitude
of the enzyme molecules in the resting state. It was found
that an average amplitude of height oscillations of P450
BM3 molecule of dimers during catalytic cycle increased
up to 5.0±2Å*s-1 that was 2.5 times larger than an average
amplitude of P450 BM3 height oscillations in the resting
state. It was obtained that the height fluctuation amplitude
of single globule of cytochrome P450 BM3 depends on
temperature, and 22˚C 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 AFM chip. In
both cases the number of enzyme molecules was ̴ 10
10
,
and the kinetics was linear during the first 10 minutes. It
was shown that in the case of solubilized enzyme k
cat
=10
-3
s-1, and in the case of immobilized enzyme k
cat
=0.4*10
-
3
s
-1
that was 2.5 times less than the first one. 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 amplitude of
enzyme globule fluctuations.
Biography
Yuri D. Ivanov was born in Alexin, Russia, in 1959. He graduated from the
Moscow Engineering Physical Institute (MEPHI) in 1982. He received his PhD
in Physics from the MEPHI in 1988 and Dr Sci. in Biol. from the Institute of
Biomedical Chemistry RAMS (Moscow) in 2000. From 2000 to present he has
been a head of laboratory of nanobiotechnology at the Institute of Biomedical
Chemistry RAMS. His current research interest is nanotechnology approaches
for the investigation of protein complexes.
yurii.ivanov@rambler.ruYuri Ivanov et al., Nano Res Appl 2017, 3:3
DOI: 10.21767/2471-9838-C1-002