(164l) Nanomechanical Property Measurement of Superoxide Dismutase Aggregates Via an Atomic Force Microscope Tip-Induced Molecular Pulling

The
application of nanotechnology in life sciences, nanobiotechnology, is the
convergence of engineering and molecular biology, leading to a new approach for
biological and chemical analysis with high sensitivity, specificity and accuracy.
In this trend, atomic force microscope (AFM) has been broadly employed in
biological study because it has no requirement of electric conductivity or
labelling process of the sample. Moreover, it can be relatively easily operated
in fluids under nearly physiological conditions. Today, as there are few alternative
experimental methods for protein imaging or molecular manipulation under physiological
condition, AFM becomes an indispensable tool for nanobiotechnology. AFM is a
versatile instrument that can image surfaces with nanoscale resolution, probe
local mechanical properties, and measure a variety of interaction forces from nN
to pN. For example, the observation of a single membrane protein,
protein–protein interaction and the mechanical unfolding of a single protein molecule
have already been reported. In addition, it has been used for bio-patterning at
the submicrometer scale as well as visualization and quantitative measurement
of materials, such as tip-induced local oxidation, dip-pen lithography. In-situ
biological detection on the patterned local area was achieved in our previous
study, which was shown to be almost impossible in other lithographic methods at
this point.

Recently,
the development of experimental tools allowing to measure of minute forces has
opened new perspectives in material- and life sciences, and the mechanical
properties of biological molecules, such as actin filaments and DNA, attracted
the interest of researchers. Especially, the capability of mechanical
manipulation on a limited area provides a new opportunity in the field of
molecular device and biophysical phenomenon. Mechanical manipulation of
selected biomolecules is a powerful approach, which has given information on
the molecular behaviours. Force spectroscopy in AFM is a relatively new
technique that analyzes the forces necessary for separating individually bound molecules
and quantifies kinetic and thermodynamic parameters of intermolecular
interactions that are unavailable by other techniques.

Here, we employed
the AFM-based molecular pulling technique to demonstrate mechanical
unfolding of Cu/Zn
superoxide dismutase (SOD1) aggregates. SOD1 is a dimeric
protein with two identical subunits arranged with eight b-stands connected by
seven loops. Each subunit contains a zinc atom (Zn) whose main role is to
stabilize the protein and a copper (Cu) atom responsible for its dismutase
activity. Over
100 mutants of SOD1 have been implicated in the neurodegenerative disease,
familial amyotrophic lateral sclerosis (FALS), such as Alzheimer's disease.
Aggregation of SOD may play a causative role on FALS and it has been reported
by a few researcher that subunits of SOD aggregation were cross-linked via
disulfide bridges, that is, tandem repeats of a single domain. Thus characterizing
mechanical properties of SOD aggregates is regarded as a meaningful approach to
comprehend structural and conformational properties of SOD aggregates.

In
this study, purified
wild type apo SOD, that is, demetallized SOD,  was diluted
with a 50 mM phosphate buffered saline
(PBS) solution (pH=7.0) at a concentration of 0.1 mM. For the purpose of oxidative
stress, 10 mM H₂O₂ and 1 mM CuSO₄ were added
to apo SOD,
dissolved
solution.
It has been extensively reported that aggregation of apo-type
protein
was readily occurred in this
condition. A formation of aggregates was investigated by UV-vis spectroscopy and these were
covalently coupled onto an amine activated Si surface. Topographic and lateral
images of immobilized aggregates were simultaneously obtained in a single scan
via an AFM, and then we obtained force distance curve by approach and retreat
of AFM tip on the aggregates. Using an Au-coated AFM tip, when tip is
brought into contact with the aggregate layer, several
aggregates bind to the AFM tip via an affinity between Au of tip and SH-residue
of free cysteine in SOD aggregates. When the tip is raised, the attached aggregates are pulled
up and away from the aggregates immobilized on the
substrate.
The
force curve was determined by measuring the deflection of the
cantilever as
it
approached and retracted from the sample.

Therefore,
we observed the morphology of immobilized aggregates and repetitive rupture
peaks of
a
multi-domain
protein aggregates
bound in parallel and series via an AFM apparatus. This technique allows one to
completely investigate the mechanical properties of SOD aggregates through a simple
mathematical calculation. Results of this study provide the mechanical property
data (base) for biomaterials and the feasibility of biomolecular-device system
via a mechanical manipulation of biological macromolecules with cells.