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Current
Pharmaceutical Biotechnology
ISSN: 1389-2010
Current Pharmaceutical Biotechnology
Volume 10, Number 5, August 2009
Contents
Single-Molecule Biophysics 2009
Guest Editor: Steven Block
Editorial Pp. 464-466
[PMID:
19689313 PubMed - indexed for MEDLINE]
Single-Molecule Force Spectroscopy Using the NanoTracker™
Optical Tweezers Platform: from Design to Application
Pp. 467-473
A. Wozniak, J. van Mameren and S.
Ragona
[Abstract] [Purchase
Article] [PMID:
19689314 PubMed - indexed for MEDLINE]
10 Years of Tension on Chromatin: Results from Single
Molecule Force Spectroscopy Pp. 474-485
F.-T. Chien and J. van Noort
[Abstract] [Purchase
Article] [PMID:
19689315 PubMed - indexed for MEDLINE]
Effect of Antibiotics and Antimicrobial
Peptides on Single Protein Motility Pp.
486-493
T. Winther and L.B. Oddershede
[Abstract] [Purchase
Article] [PMID:
19689316 PubMed - indexed for MEDLINE]
AFM Studies of λ
Repressor Oligomers Securing DNA Loops Pp.
494-501
H. Wang, L. Finzi, D.E.A. Lewis and
D. Dunlap
[Abstract] [Purchase
Article] [PMID:
19689317 PubMed - indexed for MEDLINE]
IOFF Generally Extends Fluorophore Longevity
in the Presence of An Optical Trap Pp. 502-507
J.M. Ferrer, D. Fangyuan, R.R. Brau, P.B. Tarsa
and M.J. Lang
[Abstract] [Purchase
Article] [PMID:
19689318 PubMed - indexed for MEDLINE]
Membrane Deformation at Integrin Adhesions
Pp. 508-514
E. Atilgan and B. Ovryn
[Abstract] [Purchase
Article] [PMID:
19689319 PubMed - indexed for MEDLINE]
Design Considerations for Micro- and
NanoPositioning: Leveraging the Latest for Biophysical Applications
515-521
S.C. Jordan and P.C. Anthony
[Abstract] [Purchase
Article] [PMID:
19689320 PubMed - indexed for MEDLINE]
Single-Molecule Protein Interaction Conformational
Dynamics Pp. 522-531
H.P. Lu
[Abstract] [Purchase
Article] [PMID:
19689321 PubMed - indexed for MEDLINE]
Reducing Background Contributions in
Fluorescence Fluctuation Time-Traces for Single-Molecule Measurements
in Solution Pp. 532-542
Z. Földes-Papp, S.-C.J. Liao, T. You and
B. Barbieri
[Abstract] [Purchase
Article] [PMID:
19689322 PubMed - indexed for MEDLINE]
Single-Quantum Dot Imaging with a Photon
Counting Camera Pp. 543-558
X. Michalet, R.A. Colyer, J. Antelman, O.H.W.
Siegmund, A. Tremsin, J.V. Vallerga and S. Weiss
[Abstract] [Purchase
Article] [PMID:
19689313 PubMed - indexed for MEDLINE]
Single-Molecule Fluorescence Studies
of Nucleosome Dynamics Pp. 559-568
K. Gurunathan and M. Levitus
[Abstract] [Purchase
Article] [PMID:
19689324 PubMed - indexed for MEDLINE]
Abstracts
[Back to top]
[PMID:
19689313 PubMed - indexed for MEDLINE]
Editorial: Getting High on Single Molecule Biophysics
The 5th biennial
winter workshop on Single Molecule Biophysics (SMB) was held
at the Aspen Center for Physics in Aspen, CO, over the week
January of 2-9, 2009. The resort town of Aspen lies in a high
mountain valley at 8,000 ft. (2440 m) elevation in the Rockies,
surrounded by some of the tallest peaks in Colorado, and the
stunning views from its ski slopes can literally take your
breath away. But the cognoscenti know that Aspen,
among its many other claims to fame, has also been a traditional
rendezvous point for physicists, who gather from all over
the world at the renowned Aspen Center for Physics (ACP) for
its scientific programs held in the summer and winter. In
a phrase, Aspen is to physics what Woods Hole is to biology,
with majestic mountains replacing the serene seashore. Ullr,
the Norse god of skiing, smiled once again upon the SMB meeting,
because the snow-loving conferees were treated to fresh doses
of Colorado’s famous powder nearly every day of the
week. This made for some fabulous skiing and snowboarding.
Perhaps even more fortunately, the near-daily snowstorms abated
during the two periods when it really counted, namely, as
folks were traveling into Aspen for the start of the conference
or leaving — with smiles on their faces! — at
the end. It has been my personal pleasure to organize all
five of the SMB conferences, which have been held in alternate
winters ever since 2001, and therefore to witness first-hand
the ascendancy of single molecule biophysics as a scientific
discipline. This issue of Current Pharmaceutical Biotechnology
celebrates recent progress with a special “hot topic”
volume, compiled by Editor-in-Chief Zeno Földes-Papp,
who also participated in SMB 2009 and co-authored a paper
in this issue. In these pages, you will find eleven papers
contributed by meeting participants, covering a broad range
of subjects. Taken together, they convey the current sense
of excitement and ferment in the field, and testify to the
stunning progress in single molecule research that’s
been achieved over the past decade. Today, we routinely carry
out experiments on individual biomolecules that were only
pipe dreams a scant few years ago.
The field of single molecule biophysics continues to enjoy
widespread interdisciplinary interest, solid federal funding
support, and strong growth. All in all, 110 participants were
accepted to SMB 2009 (drawn from a strong pool of nearly 200
applicants), representing an increase in enrollment of 10%
over 2007, despite the weakness in the economy. In fact, enrollment
in the SMB conferences has increased steadily every year since
these were established a decade ago. For 2009, participation
had to be limited for the first time to the number of seats
available in the auditorium at the ACP. Over the years, the
SMB conference has grown to become the premier meeting in
its field, and, as in years past, the get-together was a tremendous
success by all accounts. The SMB meetings are distinguished
by academic diversity, with participants drawn from a wide
variety of sub-disciplines, including theoretical &
experimental physics, molecular and structural biology, biochemistry,
chemistry, engineering, mathematics, and medicine. More than
50 short platform talks were presented over a five-day period.
This year also featured two jam-packed poster sessions, with
more than 75 posters. There was excellent international representation,
with participants drawn from major universities in North America,
Asia, the Middle East, and Europe. Attendees included a carefully
balanced mix of established professors, junior faculty, postdoctoral
researchers, graduate students, and representatives from national
laboratories. Approximately 23% of conferees in 2009 were
women or minorities. Financial support for the meeting was
raised from both public and private sources. This year, the
list of sponsors included Andor Inc., Chroma Inc., Cytokinetics
Inc., Hamamatsu Inc., JPK GmBH, Mad City Labs Inc., Nikon
Biomedical Inc., Physik Instrumente LLP, Princeton Instruments/Roper
Inc., the Royal Society for Chemistry (UK), Spectra-Physics
Inc., and Carl Zeiss Inc. Major funding support also came
from the National Science Foundation, which helps to underwrite
many activities of the ACP. The funding raised was primarily
used to defray a portion of the expenses of younger scientists
and those participants traveling long distances, however,
it proved possible to award at least some level of aid to
nearly every one of the participants.
In addition to intense, twice-daily science sessions and two
(crowded!) night-time poster sessions, several special events
were scheduled to enliven the proceedings. The Reception on
Sunday evening featured a performance of live bluegrass music
by some very talented Aspen-area musicians, The Flying
Dog Bluegrass Band. It was my privilege to sit in with
the band myself for a few numbers on the five-string banjo,
and also to break out my mandolin for an old-time fiddle tune
or two. Quite a few of the meeting participants, it transpired,
were long-time devotees of bluegrass music—Cees Dekker
(Technical University of Delft, Netherlands) even plays in
a European bluegrass band! —and some new converts were
won over as well.
On Wednesday, as part of its outreach program, the ACP teamed
up with a local Aspen organization to host a Physics Café
in the mezzanine lobby of Aspen’s historic Wheeler Opera
House (1889), a beautifully restored Victorian-era theater
in the heart of downtown. The Physics Café, which has
become something of a local tradition, provides an opportunity
for Aspen’s local residents to hear firsthand why the
scientists have gathered to meet and what the excitement is
all about, and to pose any questions that come to mind. These
tend to be lively events. This year, the Physics Café
featured short presentations by three of our international
participants, who courageously proceeded to field some wide-ranging
questions: Dr. Christoph Schmidt (Georg-August University,
Göttingen, Germany), Dr. Claudia Veigel (National Institute
for Medical research, Mill Hill, London, United Kingdom),
and Dr. Henrik Flyvbjerg (RISØ National Laboratory,
Roskilde, Denmark). The Physics Café was followed by
the De Wolf Lecture, held in the main theater and open to
the general public. This year, the lecture was delivered by
Prof. James A. Spudich (Stanford University), entitled “Nature's
Exquisite Nanomachines: The Dynamic and Varied City Plan of
Living Cells.” His talk was well attended, and
the local audience seemed fascinated to learn (some, for the
very first time) about the amazing array of protein-based
machines responsible for so many important processes in life,
including molecular motors such as myosin and kinesin. The
audience was sufficiently captivated that the question period
afterwards had to be extended. I can also report, as meeting
organizer, that positive feedback about the De Wolf lecture
kept pouring in for the remainder of the week, including kudos
offered by professional scientists living in the Aspen area.
Thursday afternoon featured the traditional NASTAR race, which
has been a source of friendly competition and bragging rights
at the SMB meetings since their inception. NASTAR (an abbreviation
for National Standard) is a dual-format giant slalom ski and
snowboard race where competitors can qualify for medals based
on their best race times, under a handicapping system that
pits their results against a set of uniform standards, established
nationally by elite racers at the start of each season. Biophysicists,
it seems, make for talented skiers! This year, sixty-three
meeting participants and their family members produced qualifying
times in the NASTAR race (out of roughly 75 participants),
winning a record number of medals (4 gold, 12 silver, and
20 bronze), a total that surpassed all other winter workshops
held at the ACP. The men’s ski race was won by B. Gaub
(Max Planck Inst., Munich, Germany), followed by T. Perkins
(Univ. Colorado) and A. Grindley (Yale University). The women’s
race was won by L. Finzi (Emory University), followed by C.
Grindley (Yale University) and A. Iwane (Osaka Univ., Japan).
The snowboard competition was won by E. Schaeffer (Max Planck
Institute, Dresden, Germany), followed by R. Phillips (Calif.
Inst. of Technology) and D. Rueda (Wayne State Univ.). An
award for Special Merit went to K. Frieda (Stanford Univ.),
who ran down the giant slalom course on snowshoes, edging
out J. Andrecka (Univ. Munich). Those interested in viewing
the race results for the team named “Single Molecule
Biophysics” can find these online at: http://www.nastar.com/index.jsp?pagename=raceresults&race=65105&year=2009.
Incredibly, the biophysicists managed a team ranking of 529
out of 3,917 teams in the United State for the 2008-2009 ski
season (13th percentile);
our best showing ever.
The NASTAR race was followed by the Meeting Awards Ceremony
& Banquet
that same evening, with special prizes and trophies going
to all the winners, and also to many of the losers—and
with plenty more prizes to go around for all the other meeting
participants, as well! The after-dinner ceremony also featured
the presentation of the Martin &
Beate Block Scholarship, from an endowed fund of the ACP that
furnishes a merit-based scholarship to one young scientist
selected from each of the (currently five) winter meetings
every year. The award for SMB 2009 was personally presented
by physicist and long-time Aspenite Martin Block, who was
responsible for founding the ACP winter meeting series back
in 1985, along with his wife Beate Block (who, at age 83,
also managed to snag one of the four gold NASTAR medals in
the ski race). Yes, for those of you who may be wondering,
they’re my parents! This year’s Block scholarship
was won by Ms. Anna Kochaniak, a graduate student in Antoine
van Oijen’s lab at Harvard University, for her presentation
“Single-molecule observation of the rotational and
translational movement of the PCNA sliding clamp along DNA.”
Along with a check for $500, she received a custom-made cube
of crystal glass with a three-dimensional molecular structure
laser-etched inside, displaying the PCNA clamp protein surrounding
the DNA helix.
The eleven papers found in this issue provide a snapshot of
what’s happening in the field of single molecule biophysics.
As is so often the case, progress on the technical side of
things is rapidly turned into newfound scientific knowledge.
Perhaps fittingly then, around half the contributed papers
discuss new developments in biotechnology that are driving
single molecule work, while the others present biological
results from recent research on single-molecule systems. As
the field of single molecule biophysics matures, we’re
beginning to see more commercial development of apparatus,
perhaps signaling the beginning of a trend away from the “roll
your own” approach, and one which may make single molecule
experiments more accessible those without some background
in physics or engineering. The paper by Wozniak and coworkers
describes the design and performance characteristics of one
commercial system for optical trapping, the JPK NanoTracker,
which is (to my knowledge) the first commercial system to
come equipped with the ability to sense the position of the
trapped object on the nanoscale, using quadrant photodiode
detection. As such, it should be applicable to a much wider
range of single molecule applications than the commercial
trapping systems which came before it. The paper by Jordan
& Anthony
reviews improved ways to do nanopositioning using a variety
of microprocessor-driven piezoelectric devices, an activity
that is absolutely central to work with a whole gamut of single-molecule
and single-cell approaches, including scanning-force microscopy,
ultra-high resolution optical microscopy, single-molecule
fluorescence, and optical trapping. Some of the recent progress
in single molecule biophysics has come from combining various
new imaging and measurement modalities, for example, optical
force spectroscopy and single-molecule fluorescence. However,
the marriage of these diverse techniques in a single apparatus
has posed some serious experimental challenges. For example,
the visible light emitted by a single fluorophore attached
to a protein of interest is typically about 15 orders of magnitude
less than the infrared light required to optically trap that
same protein for study, and this can make it very hard to
detect fluorescence against the background. Furthermore, the
fluorophore can rapidly become extinguished by two-photon
or other effects induced by the trapping laser. The paper
by Ferrer et al. describes a clever approach that
can substantially reduce such photobleaching, by rapidly alternating
the trapping and fluorescence excitation lasers. Michalet
and colleagues review recent progress on the development of
a new kind of low light-level camera that they are calling
“H33D”, a contrived acronym intended to mean “high
spatial-resolution, high temporal-resolution, high throughput,
three-dimensional detector.” The camera is under development
by teams of biophysicists and astrophysicists in a collaboration
that includes the Lawrence Berkeley and Livermore National
Labs; the first prototype was released in 2004. The goal is
nothing less than to produce a widefield camera that can not
only image at extremely low light levels, but also count and
time the individual photons as these arrive at each pixel
location, in effect combining the most desirable features
of sensitive avalanche photodiodes and modern EM-CCD cameras.
They report on recent test results based on recording from
single quantum dots. Földes-Papp and colleagues tackle
the thorny problem of reducing the background light contribution
in single-molecule studies, which has plagued many studies
of molecular fluorescence in solution, and particularly in
live cells. They demonstrate how optimized time-gating of
the fluorescence signal, together with time-correlated, single-photon
counting, can be used to substantially boost the experimental
signal-to-noise ratio (roughly 100-fold), making it possible
to measure analyte concentrations by correlation spectroscopy
that are as low as 15 pM. Continued progress along these lines
may eventually make it possible to record from single fluorophores
that are not otherwise immobilized, concentrated, or compartmentalized.
Six more papers report experimental results using the current
generation of single-molecule approaches. For reasons that
are doubtless obvious, a good deal of single-molecule research
has focused on the genome, and on the molecules involved carrying
out the Central Dogma of replication, transcription, and translation.
Three papers discuss revealing studies of the physical properties
of chromosomes. The paper by Chien &
van Noort reviews the state of the art on single-molecule
measurements of eukaryotic chromatin structure, using force
spectroscopy to explore the mechanical properties of individual
chromatin fibers, which involve hierarchical levels of folding
of DNA around nucleosomal cores composed of histone octamers.
The exact way that chromatin fibers get compacted (and unfolded)
has been the subject of considerable controversy, particularly
at the highest-order levels of folding, but recent results
using forces applied by optical or magnetic tweezers have
now begun to unravel some of the mysteries. This paper supplies
an excellent guide to those wishing to bone up on the subject.
A related paper by Gurunathan &
Levitus reports on the use of fluorescence to study
nucleosome dynamics, using a combination of single-molecule
FRET and fluorescence correlation spectroscopy. They find
evidence consistent with the “site exposure” model
for gene expression, where DNA can be transiently unwind from
its associated histone core (likely, in a sequence-dependent
fashion) to expose nucleic acids to the protein machinery
of the Central Dogma. Prokaryotes and even bacteriophages
have chromosomes too, of course, but these appear to be much
simpler in terms of their folding organization than those
of eukaryotes. Nevertheless, mechanical rearrangements of
the genome and associated proteins lie at the very heart of
gene expression in prokaryotes, as well as eukaryotes. The
paper by Wang et al. looks at the phenomenon of DNA
looping, which is known to regulate gene expression in bacteria
(for example, in the lac operon), and which can occur
when distant regions of DNA are brought together by bound
control proteins, such as repressors. In λ
bacteriophage, DNA looping is involved in the key decision
to become lysogenic. Wang and coworkers studied such looping
using a combination of imaging methods, including electron
microscopy and atomic force microscopy.
Single-molecule methods can also be used to study protein
mobility, or to gauge the strength of receptor-ligand interactions.
Winther &
Oddershede used an optical trap arrangement to measure the
lateral mobility of the λ
receptor in E. coli, which is found in the outer
membrane of cells. By engineering a recombinant version of
this receptor to carry a biotinylation site, they were able
to attach a streptavidin-coated bead to the extracellular
domain of the receptor, and thereby to measure its diffusional
freedom via laser light scattered light from the bead, supplied
by a low-power optical trap. They used this system to explore
the effects of various antibiotics (ampicillin, vancomycin,
and two antimicrobial peptides) on cell wall formation and
stability, assayed indirectly through the mobility of the
λ receptor.
Single molecule spectroscopy has emerged as the experimental
tool of choice for investigating protein dynamics, particularly
when trying to identify sources of temporal (or spatial) inhomogeneity
in structure and function. By studying one molecule at a time,
it not only becomes possible to reconstruct the population
(bulk) behavior, but also to learn about the population variance,
and to study any outlying (or rare) properties of potential
interest, including non-Markovian behavior. Furthermore, single
molecule spectroscopy can bypass a need for synchronized populations
of identical, prepared molecules that is so often a prerequisite
for bulk studies. The paper by Lu takes these issues up in
the specific context of structural fluctuations in the calcium
sensor, calmodulin. Finally, a contribution by Atilgan &
Ovryn examines the nucleation of integrin-based adhesion sites
on the surfaces of cells. Mature adhesion complexes help cells
to adhere to the matrix on the extracellular side of the membrane,
and are anchored to cytoskeletal elements on the cytoplasmic
face, serving to bridge the cell to its environment. The maturation
of “nouveau” adhesions, as these recruit additional
proteins and adjust their size and shape, has been a particular
matter of interest. This study used a combination of fluorescence
microscopy and a technique that the authors refer to as “phase
shifting laser feedback interferometry” to measure the
tiny distances between the ventral surfaces of cells and their
substrata, and thereby to map the membrane surface topography
with a precision of several nanometers. Although not a single-molecule
study, in the formal sense of the SMB conference, their technique
achieves a performance that beats the traditional diffraction
limit, and therefore represents one of several novel techniques
that are currently pushing the boundaries of light microscopy.
Without doubt, single molecule biophysics has a bright future
ahead, and requests have already been received for a reprise
of the meeting in 2011. We’ll have to see. The job of
organizing scientists is often compared to herding cats, which
is alternately difficult and frustrating. That said, the results
thus far have been immensely gratifying. In closing, I’d
like to acknowledge the assistance of the five grad students
from my Stanford lab who served as meeting “gophers,”
fulfilling a myriad of errands: Peter Anthony, Kirsten Frieda,
Nick Guydosh, Matt Larson, and Christian Perez, together with
the highly professional services of Ms. Jane Kelly, the Administrative
Vice President of the ACP, and her able staff, who help to
make the ACP such a special place for us scientists to gather.
Steven M. Block
Guest Editor – Current Pharmaceutical Biotechnology
Meeting Organizer, SMB 2009
S.W. Ascherman Professor of Sciences
Departments of Biology and Applied Physics
Stanford University, Stanford, CA 94305
USA
[Back to top] [Purchase
Article] [PMID:
19689314 PubMed - indexed for MEDLINE]
Single-Molecule Force Spectroscopy Using the NanoTracker™
Optical Tweezers Platform: from Design to Application
A. Wozniak, J. van Mameren and S. Ragona
Since the development of detection and analysis techniques
for optical tweezers setups, there has been an ever-increasing
interest in optical tweezers as a quantitative method, shifting
its applications from a pure manipulation tool towards the
investigation of motions and forces. With the capability of
manipulation and detection of forces of a few hundred picoNewtons
down to a fraction of a picoNewton, optical tweezers are perfectly
suitable for the investigation of single molecules. Accordingly,
the technique has been extensively used for the biophysical
characterization of biomolecules, ranging from the mechanical
and elastic properties of biological polymers to the dynamics
associated with enzymatic activity and protein motility. Here,
the use of state-of-the-art optical tweezers on the elasticity
of single DNA molecules is presented, highlighting the possibilities
this technique offers for the investigation of protein-DNA
interaction, but also for other single molecule applications.
Technical in nature, design aspects of the NanoTracker™
optical tweezers setup are addressed, presenting the recent
advances in the development of optical tweezers, ranging from
noise reduction to detection and calibration methodology.
[Back to top] [Purchase
Article] [PMID:
19689315 PubMed - indexed for MEDLINE]
10 Years of Tension on Chromatin: Results from Single Molecule
Force Spectroscopy
F.-T. Chien and J. van Noort
The compact, yet dynamic organization of chromatin plays an
essential role in regulating gene expression. Although the
static structure of chromatin fibers has been studied extensively,
the controversy about the higher order folding remains. In
the past ten years a number of studies have addressed chromatin
folding with single molecule force spectroscopy. By manipulating
chromatin fibers individually, the mechanical properties of
the fibers were quantified with piconewton and nanometer accuracy.
Here, we review the results of force induced chromatin unfolding
and compare the differences between experimental conditions
and single molecule manipulation techniques like force and
position clamps. From these studies, five major features appeared
upon forced extension of chromatin fibers: the elastic stretching
of chromatin’s higher order structure, the breaking
of internucleosomal contacts, unwrapping of the first turn
of DNA, unwrapping of the second turn of DNA, and the dissociation
of histone octamers. These events occur sequentially at the
increasing force. Resolving force induced structural changes
of chromatin fibers at the single molecule level will help
to provide a physical understanding of processes involving
chromatin that occur in vivo and will reveal the
mechanical constraints that are relevant for processing and
maintenance of DNA in eukaryotes.
[Back to top] [Purchase
Article] [PMID:
19689316 PubMed - indexed for MEDLINE]
Effect of Antibiotics and Antimicrobial Peptides on Single
Protein Motility
T. Winther and L.B. Oddershede
Following the movement of individual molecules of a bacterial
surface protein in vivo we investigated the effects
of antibiotics and antimicrobial peptides on protein motility
and membrane structure. In previous work we engineered the
λ-receptor
of Escherichia coli such that less than one receptor
per cell is in vivo biotinylated and can bind to
a streptavidin coated bead. Such a bead served as a handle
for the optical tweezers to follow the motion of an individual
receptor. In an un-perturbed living cell the λ-receptor
performs a confined diffusive motion. The λ-receptor
links to the peptidoglycan layer, and indeed, a perturbation
of the peptidoglycan layer had a pronounced effect on the
motility of the receptor: The motility significantly decreases
upon treatment with vancomycin or ampicillin, to study the
effect of vancomycin we used strains with increased membrane
permeability. As the motility of an individual receptor was
monitored over an extended amount of time we were able to
observe a temporal evolution of the action of vancomycin.
Antimicrobial peptides (AMPs) are alternatives to conventional
antibiotics in the treatment of bacterial infections. Therefore,
we also investigated the effect of the toxic AMP polymyxin
B (PMB) which targets both the outer and inner membranes and
kills the organism. PMB significantly decreased the motility
of the λ-receptor.
On the basis of these findings we confirm that the λ-receptor
is firmly attached to the peptidoglycan layer, and that an
antibiotic or AMP mediated destruction of the dynamic peptidoglycan
synthesis decreases the receptor motion.
[Back to top] [Purchase
Article] [PMID:
19689317 PubMed - indexed for MEDLINE]
AFM Studies of λ
Repressor Oligomers Securing DNA Loops
H. Wang, L. Finzi, D.E.A. Lewis and D. Dunlap
Large, cooperative assemblies of proteins that wrap and/or
loop genomic DNA may “epigenetically” shift configurational
equilibria that determine developmental pathways. Such is
the case of the λ
bacteriophage which may exhibit virulent (lytic) or quiescent
(lysogenic) growth. The lysogenic state of λ
prophages is maintained by the λ
repressor (CI), which binds to tripartite operator sites in
each of the OL
and OR control regions
located about 2.3 kbp apart on the phage genome and represses
lytic promoters. Dodd and collaborators have suggested that
an initial loop formed by interaction between CI bound at
OR and
OL provides
the proper scaffold for additional CI binding to attenuate
the PRM
promoter and avoid over production of CI. Recently, the looping
equilibrium as a function of CI concentration was measured
using tethered particle motion analysis, but the oligomerization
of CI in looped states could not be determined. Scanning force
microscopy has now been used to probe these details directly.
An equilibrium distribution of looped and unlooped molecules
confined to a plane was found to be commensurate to that for
tethered molecules in solution, and the occupancies of specific
operator sites for several looped and unlooped conformations
were determined. Some loops appeared to be sealed by oligomers
of 6-8, most by oligomers of 10-12, and a few by oligomers
of 14-16.
[Back to top] [Purchase
Article] [PMID:
19689318 PubMed - indexed for MEDLINE]
IOFF Generally Extends Fluorophore Longevity in the Presence
of An Optical Trap
J.M. Ferrer, D. Fangyuan, R.R. Brau, P.B. Tarsa
and M.J. Lang
The combination of optical tweezers force microscopy
and single molecule fluorescence has previously been complicated
by trap-induced photobleaching. Recent studies have suggested
that this effect is caused by a sequential absorption of photons,
leading to ionization of the fluorescent singlet state. In
this work, we show the range of effects of optical trapping
radiation on common fluorescent dyes. Using the interlaced
optical force fluorescence (IOFF) laser modulation technique,
we show that the removal of simultaneous near infrared radiation
dramatically reduces photobleaching effects. However, these
studies show that the sequential addition of near infrared
radiation in some cases extends photobleaching longevity beyond
the natural intrinsic decay. We suggest a refined photoelectronic
mechanism that accounts for the possibility of reverse intersystem
crossing from a reactive triplet state and explains the nature
of trap-induced photobleaching.
[Back to top] [Purchase
Article] [PMID:
19689319 PubMed - indexed for MEDLINE]
Membrane Deformation at Integrin Adhesions
E. Atilgan and B. Ovryn
In order to measure the nucleation of nouveau adhesions
on the ventral surface of a cell, we have combined phase shifting
laser feedback interferometry with a high numerical aperture
inverted fluorescence microscope. We use fluorescence to image
molecules at the adhesion site and stage scanning interference
microscopy in order to measure the distance between the ventral
surface of a cell and the substratum with several nanometer
precision. Our analytic and Monte Carlo simulations of integrin
mediated adhesions predict several features of these nouveau
adhesions. An analysis of the energetics of membrane bending
and the effects of a composite system of freely diffusing
repellers and receptors and a fixed network of ligands on
the extracellular matrix predicts that a small bundle of actin
filaments should be able to push the membrane down to the
extracellular matrix and nucleate a nouveau adhesion with
critical radius below the diffraction limit. We have obtained
a map of the reflectivity of the ventral surface of fixed
metastatic mammary adenocarcinoma cells and we have shown
that the data are correlated with markers for a focal adhesion
adaptor protein. We are modeling the interference of the incident
electric field with the field reflected from the ventral surface
so as to obtain the surface topography at focal adhesions
from the optical phase data.
[Back to top] [Purchase
Article] [PMID:
19689320 PubMed - indexed for MEDLINE]
Design Considerations for Micro- and NanoPositioning: Leveraging
the Latest for Biophysical Applications
S.C. Jordan and P.C. Anthony
Biophysical applications ranging from fluorescence microassays
to single-molecule microscopy are increasingly dependent on
automated nanoscale positional control and stability. A whirlwind
of motion-industry innovation has resulted in an array of
new motion options offering significant improvements in application
performance, reproducibility and throughput. The challenge
to leverage these developments depends on researchers, engineers
and motion vendors acquiring a common language of specifications
and a shared understanding of the challenges posed by application
needs.
To assist in building this shared understanding, this article
reviews today’s motion technologies, beginning with
a concise review of key principles of motion control focusing
on applications. It progresses through illustrations of sensor/encoder
technologies and servo techniques. A spectrum of classical
and recent motion technologies is explored, from stepper and
servo actuation of conventional microscopy stages, to advanced
piezo stack nanopositioners capable of picometer precision,
to novel ultrasonic resonant piezomotors and piezo-ceramic-based
mechanisms capable of high-force positioning over many millimeters
while providing resolutions down into the sub-nanometer range.
A special emphasis is placed on the effects of integrating
multiple motion technologies into an application, such as
stacking a fine nanopositioner atop a long-travel stage. Examples
and data are presented to clarify these issues, including
important and insightful new stability measurements taken
directly from an advanced optical trapping application. The
important topics of software and interfacing are also explored
from an applications perspective, since design-and-debugging
time, synchronization capabilities and overall throughput
are heavily dependent on these often-overlooked aspects of
motion system design.
The discussion is designed to illuminate specifications-related
topics that become increasingly important as precision requirements
tighten. Throughout, both traditional and novel techniques
and approaches are explored so that readers are left with
a solid overview of the state of the art, and an actionable
perspective that readies them to discuss and evaluate specifications
and vendor capabilities against practical application requirements.
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Article] [PMID:
19689321 PubMed - indexed for MEDLINE]
Single-Molecule Protein Interaction Conformational
Dynamics
H.P. Lu
Protein conformational fluctuations and dynamics, often associated
with static and dynamic inhomogeneities, play a crucial role
in biomolecular functions. It is extremely difficult to characterize
such spatially and temporally inhomogeneous dynamics in an
ensemble-averaged measurement, especially when the proteins
involve in a multiple-step and multiple-conformation complex
chemical interactions and transformations, such as in protein-protein
interactions and protein-DNA interactions. Single-molecule
spectroscopy is a powerful approach to analyze protein conformational
dynamics under physiological conditions, providing dynamic
perspectives on a molecular-level understanding of protein
structure-function mechanisms.
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[Purchase
Article] [PMID:
19689322 PubMed - indexed for MEDLINE]
Reducing Background Contributions in Fluorescence
Fluctuation Time-Traces for Single-Molecule Measurements in
Solution
Z. Földes-Papp, S.-C.J. Liao, T. You and
B. Barbieri
We first report on the development of new microscope
means that reduce background contributions in fluorescence
fluctuation methods: i) excitation shutter, ii) electronic
switches, and iii) early and late time-gating. The elements
allow for measuring molecules at low analyte concentrations.
We first found conditions of early and late time-gating with
time-correlated single-photon counting that made the fluorescence
signal as bright as possible compared with the fluctuations
in the background count rate in a diffraction-limited optical
set-up. We measured about a 140-fold increase in the amplitude
of autocorrelated fluorescence fluctuations at the lowest
analyte concentration of about 15 pM, which gave a signal-to-background
advantage of more than two-orders of magnitude. The results
of this original article pave the way for single-molecule
detection in solution and in live cells without immobilization
or hydrodynamic/electrokinetic focusing at longer observation
times than are currently available.
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[Purchase
Article] [PMID:
19689323 PubMed - indexed for MEDLINE]
Single-Quantum Dot Imaging with a Photon Counting Camera
X. Michalet, R.A. Colyer, J. Antelman, O.H.W.
Siegmund, A. Tremsin, J.V. Vallerga and S. Weiss
The expanding spectrum of applications of single-molecule
fluorescence imaging ranges from fundamental in vitro
studies of biomolecular activity to tracking of receptors
in live cells. The success of these assays has relied on progress
in organic and non-organic fluorescent probe developments
as well as improvements in the sensitivity of light detectors.
We describe a new type of detector developed with the specific
goal of ultra-sensitive single-molecule imaging. It is a wide-field,
photon-counting detector providing high temporal and high
spatial resolution information for each incoming photon. It
can be used as a standard low-light level camera, but also
allows access to a lot more information, such as fluorescence
lifetime and spatio-temporal correlations. We illustrate the
single-molecule imaging performance of our current prototype
using quantum dots and discuss on-going and future developments
of this detector.
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[Purchase
Article] [PMID:
19689324 PubMed - indexed for MEDLINE]
Single-Molecule Fluorescence Studies of Nucleosome Dynamics
K. Gurunathan and M. Levitus
Single-molecule techniques have transformed dramatically the
way we think about biophysics, making it possible to address
questions about the dynamics of systems in equilibrium that
were practically unthinkable just a decade ago. This review
focuses on how single-molecule fluorescence and fluorescence
correlation techniques have allowed the investigation of the
mechanisms by which nucleosomes allow enzymes and other proteins
to access DNA regions that are buried within the nucleosome
structure. It has been established that DNA-protein and protein-protein
interactions in nucleosomes are very dynamic. The dynamics
of the interactions between the DNA and the histone proteins
have been investigated by single-molecule FRET and fluorescence
correlation spectroscopy. Results are consistent with the
so-called site exposure model, in which DNA transiently and
spontaneously unwraps from the histone core. DNA accessibility
is greatest for sites close to the DNA termini, and decreases
towards the nucleosome dyad. Evidence also suggests that DNA
sequence plays an important role. The dynamics of the H2A-H2B
dimers within the nucleosome has also been addressed by several
groups in terms of their implications in determining nucleosome
stability and DNA dynamics.
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