He, Chengzhi

Hu, Chunguang

Hu, Xiaodong

Hu, Xiaotang

Xiao, Adam

Li, Hongbin

Ajuntament de Barcelona

Repositori Obert de Coneixement de l'Ajuntament de Barcelona

Gaceta municipal de Barcelona. Año 043, nº 48 (26 nov. 1956)

Elsevier - Publisher Connector 

Tuesday, February 10, 2015 355aPlatform: Force Spectroscopy and Scanning
Probe Microscopy
1780-Plat
Imaging and Three-Dimensional Reconstruction of Chemical Groups in a
Protein Complex using DNA Labels
Duckhoe Kim1, Ozgur Sahin1,2.
1Biological Sciences, Columbia University, New York, NY, USA, 2Physics,
Columbia University, New York, NY, USA.
Atomic force microscopy (AFM) is a powerful tool for imaging and chemical
characterization of bio-samples at molecular resolution in physiologically-
relevant environments. However, the localized tip-sample interactions limit
high-resolution images to the topmost layer of surfaces. Consequently, charac-
terizing the three-dimensional (3-D) inner structures of molecules has been a
challenge. Here, we demonstrate three-dimensional (3-D) localization of chem-
ical groups within a single protein complex using AFM.We employ short DNA
sequences to label specific chemical groups inside a protein complex. T-shaped
cantilevers functionalized with complementary probe DNAs allow locating
each label with sequence specificity and sub-nanometer resolution. We also
measure pairwise distances between labels and reconstruct the 3-D loci of
the target groups using simple geometric calculations. Experiments with the
biotin-streptavidin complex showed that the 3-D loci of carboxylic acids of bi-
otins are within 2-Angstroms of their respective 3-D loci in the corresponding
crystal structure, suggesting AFM may complement existing structural biolog-
ical techniques in solving structures that are difficult to study due to their size
and complexity. This technique maybe finds applications in studying structure
of DNA or RNA binding proteins.
1781-Plat
Acoustic Force Spectroscopy
Douwe Kamsma1, Gerrit Sitters1, Gregor Thalhammer2,
Monika Ritsch-Marte2, Erwin J.G. Peterman1, Gijs J.L. Wuite1.
1Physics and astronomy, VU University Amsterdam, Amsterdam,
Netherlands, 2Department fu¨r Physiologie und Medizinische Physik,
Medizinische Universita¨t Innsbruck, Innsbruck, Austria.
Single-molecule force spectroscopy has become an indispensable tool to unravel
the structural and mechano-chemical properties of biomolecules. In most force-
spectroscopy instruments only a limited number of biomolecules can be studied
simultaneously, which reduces experimental throughput and limits statistics. At
the same time, many independent measurements are required to distinguish the
intrinsic stochasticity of the process of interest from heterogeneity.
With Acoustic Force Spectroscopy (AFS) we extend the force-spectroscopy
toolbox with an acoustic manipulation device that allows exerting acoustic
forces on tethered molecules. AFS is a Lab-on-a-chip device consisting of a
flow cell of two glass plates with a fluid chamber in between and a piezo element
glued on top. While applying an alternating voltage to the piezo element, forces
from sub-pN to hundreds of pNs are exerted to thousands of biomolecules in par-
allel, with sub-millisecond response time and inherent stability.
As a proof of concept we performed force-extensionmeasurements on DNA and
RecA-coated DNA. These experiments demonstrate that AFS can be used to
apply highly controlled forces up to at least 120 pN, with a force ramp speed be-
tween 104 - 102 pN/s and showing inherent stability over tens of hours over an
observation area of at least 1mm2.AFSexperiments are highly parallel, allowing
the simultaneous measurement of thousands of biomolecules simultaneously, in
a single field of view.Wedemonstrate the use of this bymapping the energy land-
scape of the DIG/anti-DIG antibody-antigen bond over 6 orders of magnitude of
force loading rates within 2 days of experimentation.
AFS distinguishes itself by its relative simplicity, low cost and compactness,
which allow straightforward implementation in lab-on-a-chip devices. These
aspects will help to spread single-molecule methods from the realm of funda-
mental research in specialized laboratories towards more wide-spread applica-
tions in for example molecular biology and medical diagnostics.
1782-Plat
Revisiting the Free Energy of Modular Proteins under Force
Ionel Popa1, Jaime Andre´s Rivas-Pardo1, Edward C. Eckels1,
Jessica Valle-Orero1, Thomas B. Kahn1, Ronen Berkovich2,
Guillaume Stirnemann3, Hu Chen4, Vicente I. Fernandez5,
Bruce J. Berne3, Jie Yan6, Julio M. Fernandez1.
1Biological Sciences, Columbia University, New York, NY, USA, 2Chemical
Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel,
3Chemistry, Columbia University, New York, NY, USA, 4Physics, Xiamen
University, Xiamen, China, 5Civil and Environmental Engineering,
Massachusetts Institute of Technology, Cambridge, MA, USA, 6Physics,
National University of Singapore, New York, Singapore.The elasticity of muscle tissue relies on tandem modular proteins such as titin,
which gives muscles passive elasticity. A free-energy model is the only way to
understand and predict the behavior under force of such complex proteins,
composed of hundreds of individual domains. Here we use magnetic tweezers
to measure the dynamics of single tandem modular proteins under constant
force conditions during hour-long recordings. At forces between 4 to 17 pN
we measure unfolding/refolding of individual domains as upward/downward
steps in the end-to-end protein length, while higher forces yield unfolding steps
exclusively. We find a strong force dependency of the step size of proteins un-
dergoing folding/unfolding reactions with the applied force. This finding con-
tradicts current free energy models of proteins that typically do not consider
the polymeric nature of a denatured polypeptide chain under force and simply
scale the free energy of a protein with the mechanical work. To explain the
measured step size dependency with force we propose a new free energy model
that also considers the entropic work needed to extend the molecule. Brownian
dynamics simulations over the proposed free energy landscape accurately
reproduce our experimental benchmarks. The experimental and theoretical ad-
vances demonstrated in this work provide a novel view on the free energy of
proteins under force, now permitting a more realistic modeling of tissue
elasticity.
1783-Plat
Directly Observing the Reversible Unfolding and Refolding of an Alpha/
Beta Protein by Single-Molecule Atomic Force Microscopy
Chengzhi He1, Chunguang Hu2, Xiaodong Hu2, Xiaotang Hu2, Adam Xiao1,
Hongbin Li1,2.
1Chemistry, The University of British Columbia, Vancouver, BC, Canada,
2State Key Laboratory of Precision Measurements Technology and
Instruments, Tianjin University, Tianjin, China.
Single-molecule force spectroscopy based on atomic force microscopy (AFM)
has evolved into a powerful tool to study protein folding-unfolding dynamics
and mechanism. Direct observation of protein refolding in real time using
AFM has been challenging. Proteins that have been observed to refold using
AFM are mainly limited to all-alpha proteins, such as ankyrin and calmodulin.
Here we report the use of AFM to directly monitor the folding of an alpha/beta
protein, NuG2. Our results indicate that at slow pulling speeds (<50 nm/s), the
refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces
the difference between the unfolding forces and refolding forces, bringing the
non-equilibrium unfolding-refolding towards equilibrium. At very slow pulling
speeds (~2 nm/s), reversible unfolding and refolding were observed. Based on
Crooks Theorem, we measured the equilibrium free energy change between the
folded and unfolded states of NuG2, which is in good agreement with values
reported using bulk chemical denaturation method. Our results demonstrate
the utility of AFM in elucidating the unfolding-refolding dynamics of proteins
close to equilibrium.
1784-Plat
Electromagnetic Tweezers with Independent Force and Torque Control
Chang Jiang, Troy A. Lionberger, Diane M. Wiener, Edgar Meyho¨fer.
University of Michigan, Ann Arbor, MI, USA.
Mechanical forces and torques play an important role in many biological pro-
cesses. A range of instruments have been developed for direct and precise mea-
surements of force and torque applied by biomolecules ranging from proteins to
nucleic acids. However, simultaneous force and torque applications in which
the two components are decoupled and controlled independently remain chal-
lenging and have only been implemented by recently-developed hybrid mag-
netic tweezers combining electro- and permanent magnets. Our goal was to
develop bona fide electromagnetic tweezers (eMT) that can apply force and tor-
que on single biomolecules or polymer molecules conjugated via superpara-
magnetic microspheres and can control the two components independently
simply by changing the currents applied to different coils of the eMT. We im-
plemented our eMT by combining a monopole that generates a force and a set
of quadrupoles that generate a torque. To demonstrate the capability of our
tweezers we attached Janus beads to single DNA molecules. We show that ten-
sion in the piconewton force range can be applied to single DNAmolecules and
simultaneously the molecule can be twisted with torques in the piconewton-
nanometer regime. Our results also demonstrate that the two components are
independently controlled. At various force levels applied to the Janus bead,
the trap torsional stiffness can be changed simply by varying the current magni-
tude applied to the quadrupole torque tweezers. Our eMT voids the need of
complex positioners to translate and rotate permanent magnets or frequency
modulation of current to decouple the force and torque used in previous works.
We believe that the flexible control of the mechanics of biomolecules with our
eMT will enable novel studies of DNA-protein interactions and DNA confor-
mation dynamics.
