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 th Read More

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 a Read More

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 mechanic Read More

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 tweez Read More

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 regio Read More

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 Read More

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 Read More

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 de Read More

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, s Read More

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 fluore Read More

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 Read More

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 ot Read More