cells. The large size of these molecules imposes certain limitations
on the information attainable from bulk measurements, because the
macromolecules in a population can have diverse conformations and
-react differently to external stimuli. The individualized, and
-sometimes rare, behaviors of macromolecules can have important
-implications for their functions inside the cell. Recently developed
-single molecule techniques, in which these macromolecules are studied
-one at a time, have provided important and complementary information
-about the functional mechanisms of several biological
-systems\citep{bustamante08}.
+behaviors. The bulk measurement averages over these differences,
+producing excelent statistics on the mean, but making it difficult to
+understand the variation. The individualized, and sometimes rare,
+behaviors of macromolecules can have important implications for their
+functions inside the cell\cite{TODO}. Single molecule techniques, in
+which the macromolecules are studied one at a time, allow direct
+access to the variation within the population without averaging. This
+provides important and complementary information about the functional
+mechanisms of several biological systems\citep{bustamante08}.
% why AFM & what an AFM is
Single molecule techniques for the study of biological macromolecules
include optical measurements, \ie, single molecule fluorescence
microscopy and spectroscopy, and mechanical manipulations of
individual macromolecules, \ie, force microscopy and spectroscopy
-using atomic force microscopes (AFM), laser tweezers\citep{forde02},
-magnetic tweezers\citep{smith92}, or biomembrane force
-probes\citep{merkel99}. Of these mechanical manipulation methods, AFM
-is the most widely used due to the availability of user-friendly
-commercial instruments. AFM has been employed on several types of
-biological macromolecules, such as mechanically unfolding
+using atomic force microscopes (AFMs), laser tweezers\citep{forde02},
+magnetic tweezers\citep{smith92}, biomembrane force
+probes\citep{merkel99}, and centrifugal
+microscopes\cite{halvorsen2010}. Of these mechanical manipulation
+methods, AFM is the most widely used due to the availability of
+user-friendly commercial instruments. AFM has been employed on
+several types of biological macromolecules, mechanically unfolding
proteins\citep{carrion-vazquez99b} and forcing structural transitions
in DNA\citep{rief99} and polysaccharides\citep{rief97a}. An AFM uses
a sharp tip integrated at the end of a cantilever to interact with the
sample. Cantilever bending is measured by a laser reflected off the
-cantilever and incident on a position sensitive photodetector. When
-the bending force constant of the cantilever is known\citep{levy02},
-the force applied to the sample can be calculated. The forces that
-can be applied and measured with an AFM range from tens of piconewtons
-to hundreds of nanonewtons. The investigation of the unfolding and
-refolding processes of individual protein molecules by the AFM is
-feasible because many globular proteins unfold under external forces
-in this range. Since elucidating the mechanism of protein folding is
-currently one of the most important problems in biological sciences,
-the potential of the AFM for revealing significant and unique
-information about protein folding has stimulated much effort in both
-experimental and theoretical research.
+cantilever and incident on a position sensitive photodetector
+(\cref{fig:afm-schematic}. When the bending force constant of the
+cantilever is known\citep{levy02}, the force applied to the sample can
+be calculated. The forces that can be applied and measured with an
+AFM range from tens of piconewtons to hundreds of nanonewtons. The
+investigation of the unfolding and refolding processes of individual
+protein molecules by the AFM is feasible because many globular
+proteins unfold under external forces in this range. Since
+elucidating the mechanism of protein folding is currently one of the
+most important problems in biological sciences, the potential of the
+AFM for revealing significant and unique information about protein
+folding has stimulated much effort in both experimental and
+theoretical research.
+
+\begin{figure}
+ \begin{center}
+ \includegraphics{figures/schematic/afm}%
+ \label{fig:afm-schematic}}
+ \caption{(a) Operating principle for Atomic Force Microscopy. A
+ sharp tip integrated at the end of a cantilever interacts with the
+ sample. Cantilever bending is measured by a laser reflected off
+ the cantilever and incident on a position sensitive
+ photodetector.}
+ \end{center}
+\end{figure}
% AFM unfolding procedure
In a mechanical unfolding experiment, a protein polymer is tethered
between two surfaces: a flat substrate and an AFM tip. The polymer is
stretched by increasing the separation between the two surfaces
-(\cref{fig:schematic}). The most common mode is the constant speed
-experiment in which the substrate surface is moved away from the tip
-at a uniform rate. The tethering surfaces, \ie, the AFM tip and the
-substrate, have much larger radii of curvature than the dimensions of
-single domain globular proteins that are normally used for folding
-studies. This causes difficulties in manipulating individual protein
-molecules because nonspecific interactions between the AFM tip and the
-substrate may be stronger than the forces required to unfold the
-protein when the surfaces are a few nanometers apart. To circumvent
-these difficulties, globular protein molecules are linked into
-polymers, which are then used in the AFM
+(\cref{fig:unfolding-schematic}). The most common mode is the
+constant speed experiment in which the substrate surface is moved away
+from the tip at a uniform rate. The tethering surfaces, \ie, the AFM
+tip and the substrate, have much larger radii of curvature than the
+dimensions of single domain globular proteins that are normally used
+for folding studies. This causes difficulties in manipulating
+individual protein molecules because nonspecific interactions between
+the AFM tip and the substrate may be stronger than the forces required
+to unfold the protein when the surfaces are a few nanometers apart.
+To circumvent these difficulties, globular protein molecules are
+linked into polymers, which are then used in the AFM
studies\citep{carrion-vazquez99b,chyan04,carrion-vazquez03}. When
such a polymer is pulled from its ends, each protein molecule feels
the externally applied force, which increases the probability of
individual unfolding events are separated from each other in space and
time, facilitating single molecule studies.
+\begin{figure}
+ \begin{center}
+ \subfloat[][]{\includegraphics{figures/schematic/unfolding}%
+ \label{fig:unfolding-schematic}}
+ % \hspace{.25in}%
+ \subfloat[][]{\includegraphics{figures/expt-sawtooth/fig}%
+ \label{fig:expt-sawtooth}}
+ \caption{(a) Schematic of the experimental setup for mechanical
+ unfolding of proteins using an AFM (not to scale). An experiment
+ starts with the tip in contact with the substrate surface, which
+ is then moved away from the tip at a constant speed. $x_t$ is the
+ distance traveled by the substrate, $x_c$ is the cantilever
+ deflection, $x_u$ is the extension of the unfolded polymer, and
+ $x_f=x_{f1}+x_{f2}$ is the extension of the folded polymer. (b)
+ An experimental force curve from stretching a ubiquitin polymer
+ with the rising parts of the peaks fitted to the WLC
+ model\citep{chyan04}. The pulling speed used was $1\U{$\mu$m/s}$.
+ The irregular features at the beginning of the curve are due to
+ nonspecific interactions between the tip and the substrate
+ surface, and the last high force peak is caused by the detachment
+ of the polymer from the tip or the substrate surface. Note that
+ the abscissa is the extension of the protein chain $x_t-x_c$.}
+ \end{center}
+\end{figure}
+
% AFM unfolding analysis, what we'll do.
Much theoretical and computational work has been done in order to
extract information about the structural, kinetic, and energetic
the absence of force as well as the distance from the native state to
the transition state along the pulling direction. The Monte Carlo
simulation method has been used since the first report of mechanical
-unfolding experiment using AFM\citep{rief97b},
-%,rief97a,rief98,carrion-vazquez99b,best02,zinober02,jollymore09},
-however, a comprehensive description and discussion of the simulation
-procedures and the intricacies involved has not been reported. In
-this paper, we provide a detailed description of the simulation
-procedure, including the theories, approximations, and assumptions
-involved. We also explain the procedure for extracting kinetic
-properties of the protein from experimental data and introduce a
-quantitative measure of fit quality between simulation and
-experimental results. In addition, the effects of various
-experimental parameters on force curve appearance are demonstrated,
-and the errors associated with different methods of data pooling are
-discussed. We believe that these results will be useful in
-experimental design, artifact identification, and data analysis for
-single molecule mechanical unfolding experiments.
-
-\begin{figure}
-\begin{center}
-\subfloat[][]{\includegraphics{figures/schematic/unfolding}\label{fig:schematic}}
-%\hspace{.25in}%
-\subfloat[][]{\includegraphics{figures/expt-sawtooth/fig}\label{fig:expt-sawtooth}}
-\caption{(a) Schematic of the experimental setup for mechanical
- unfolding of proteins using an AFM (not to scale). An experiment
- starts with the tip in contact with the substrate surface, which is
- then moved away from the tip at a constant speed. $x_t$ is the
- distance traveled by the substrate, $x_c$ is the cantilever
- deflection, $x_u$ is the extension of the unfolded polymer, and
- $x_f=x_{f1}+x_{f2}$ is the extension of the folded polymer. (b) An
- experimental force curve from stretching a ubiquitin polymer with
- the rising parts of the peaks fitted to the WLC
- model\citep{chyan04}. The pulling speed used was $1\U{$\mu$m/s}$.
- The irregular features at the beginning of the curve are due to
- nonspecific interactions between the tip and the substrate surface,
- and the last high force peak is caused by the detachment of the
- polymer from the tip or the substrate surface. Note that the
- abscissa is the extension of the protein chain $x_t-x_c$.}
-\end{center}
-\end{figure}
+unfolding experiment using
+AFM\citep{rief97a,rief97b,rief98,carrion-vazquez99b,best02,zinober02,jollymore09},
+however...
}
@article{jones05,
+author = "R.E. Jones and D.P. Hart",
title = "Force interactions between substrates and {SPM} cantilevers
immersed in fluids",
journal = "Tribology International",
pages = "355--361",
year = 2005,
issn = "0301-679X",
-author = "R.E. Jones and D.P. Hart",
keywords = "AFM",
keywords = "Liquid",
keywords = "Hydrodynamic",
doi = "DOI: 10.1016/j.triboint.2004.08.016",
url = "http://www.sciencedirect.com/science/article/B6V57-4DN9K7J-1/2/fef91ac022594c2c6a701376d83ecd31",
}
+
+@Article {halvorsen2010,
+author = "Ken Halvorsen and Wesley P. Wong",
+title = "Massively parallel single-molecule manipulation using centrifugal force",
+journal = "arXiv",
+year = 2009,
+abstract = {Precise manipulation of single molecules has already led
+ to remarkable insights in physics, chemistry, biology and
+ medicine. However, widespread adoption of single-molecule techniques
+ has been impeded by equipment cost and the laborious nature of
+ making measurements one molecule at a time. We have solved these
+ issues with a new approach: massively parallel single-molecule force
+ measurements using centrifugal force. This approach is realized in a
+ novel instrument that we call the Centrifuge Force Microscope (CFM),
+ in which objects in an orbiting sample are subjected to a
+ calibration-free, macroscopically uniform force-field while their
+ micro-to-nanoscopic motions are observed. We demonstrate
+ high-throughput single-molecule force spectroscopy with this
+ technique by performing thousands of rupture experiments in
+ parallel, characterizing force-dependent unbinding kinetics of an
+ antibody-antigen pair in minutes rather than days. Additionally, we
+ verify the force accuracy of the instrument by measuring the
+ well-established DNA overstretching transition at 66 $\pm$ 3
+ pN. With significant benefits in efficiency, cost, simplicity, and
+ versatility, "single-molecule centrifugation" has the potential to
+ revolutionize single-molecule experimentation, and open access to a
+ wider range of researchers and experimental systems.},
+url = "http://arxiv.org/abs/0912.5370",
+}
projects / thesis.git / commitdiff