Current Pharmaceutical Biotechnology - Volume 10, Issue 5, 2009

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 Foldes-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 Cafe 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 Cafe, 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 Cafe featured short presentations by three of our international participants, who courageously proceeded to field some wide-ranging questions: Dr. Christoph Schmidt (Georg-August University, Gottingen, Germany), Dr. Claudia Veigel (National Institute for Medical research, Mill Hill, London, United Kingdom), and Dr. Henrik Flyvbjerg (RISO National Laboratory, Roskilde, Denmark). The Physics Cafe 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.

Since the development of detection and analysis techniques for optical tweezers setups, there has been an everincreasing 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.

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.

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.

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.

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.

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.

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.

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 structurefunction mechanisms.

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.

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.

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.