Explore the vast range of titles available.

Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis.

Accurate force fields are essential for the success of molecular dynamics simulations. In apparent contrast to the conformational preferences of most force fields, recent NMR experiments suggest that short polyalanine peptides in water populate the polyproline II structure almost exclusively. To investigate this apparent contradiction, with its ramifications for the assessment of molecular force fields and the structure of unfolded proteins, we performed extensive simulations of Ala5 in water (∼5μs total time), using twelve different force fields and three different peptide terminal groups. Using either empirical or density-functional-based Karplus relations for the J-couplings, we find that most current force fields do overpopulate the α-region, with quantitative results depending on the choice of Karplus relation and on the peptide termini. Even after reweighting to match experiment, we find that Ala5 retains significant α- and β-populations. In fact, several force fields match the experimental data well before reweighting and have a significant helical population. We conclude that radical changes to the best current force fields are not necessary, based on the NMR data. Nevertheless, experiments on short peptides open the way toward the systematic improvement of current simulation models.

All-atom molecular dynamics (MD) simulations of protein folding allow analysis of the folding process at an unprecedented level of detail. Unfortunately, such simulations have not yet reached their full potential both due to difficulties in sufficiently sampling the microsecond timescales needed for folding, and because the force field used may yield neither the correct dynamical sequence of events nor the folded structure. The ongoing study of protein folding through computational methods thus requires both improvements in the performance of molecular dynamics programs to make longer timescales accessible, and testing of force fields in the context of folding simulations. We report a ten-microsecond simulation of an incipient downhill-folding WW domain mutant along with measurement of a molecular time and activated folding time of 1.5 microseconds and 13.3 microseconds, respectively. The protein simulated in explicit solvent exhibits several metastable states with incorrect topology and does not assume the native state during the present simulations.

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.

The Saffman-Delbrück approximation is commonly used in biophysics to relate the membrane inclusion size to its translational diffusion coefficient and membrane viscosity. However, this approximation has a restricted validity range, and its application to determination of inclusion sizes from diffusion data may in certain cases lead to unreliable results. At the same time, the model by Hughes et al. (Hughes, B. D., B. A. Pailthorpe, and C. R. White. 1981. J. Fluid Mech. 110:349–372.), providing diffusion coefficients of membrane inclusions for arbitrary inclusion sizes and viscosities of the membrane and surrounding fluids, involves substantial computational efforts, which prevents its use in practical data analysis. We develop a simple and accurate analytical approximation to the Hughes et al. model and demonstrate its performance and utility by applying it to the recently published experimental data on translational diffusion of micrometer-sized membrane domains.

Free energy perturbation calculations are carried out to estimate the effective pKa of an arginine (Arg) sidechain as a function of its location in the dipalmitoylphosphatidylcholine bilayer. Similar to previous all-atom simulations of the voltage sensor domain of a potassium channel in the membrane with charged Arg residues, the membrane and water structures deform to stabilize the charge of the Arg analog. As a result, the computed pKa is >7 throughout the membrane although the value is very close to 7 near the center of the bilayer. With additional stabilizations from negatively charged amino acids or lipid molecules, it is reasonable to expect that Arg in membrane proteins (once in the membrane) can adopt the protonated state despite the low dielectric nature of the bulk lipid membrane.

The p53 protein, a transcription factor of key importance in tumorigenesis, is suggested to diffuse one-dimensionally along DNA via its C-terminal domain, a process that is proposed to regulate gene activation both positively and negatively. There has been no direct observation of p53 moving along DNA, however, and little is known about the mechanism and rate of its translocation. Here, we use single-molecule techniques to visualize, in real time, the one-dimensional diffusion of p53 along DNA. The one-dimensional diffusion coefficient is measured to be close to the theoretical limit, indicative of movement along a free energy landscape with low activation barriers. We further investigate the mechanism of translocation and determine that p53 is capable of sliding—moving along DNA while in continuous contact with the duplex, rather than through a series of hops between nearby bases.

X-ray data are presented for the benchmark dipalmitoylphosphatidylcholine lipid bilayer in the most biologically relevant state in which the bilayers are fully hydrated and in the fluid (liquid-crystalline) phase. Form factors F( q z) are obtained from a combination of two sample preparations, oriented stacks of bilayers for q z extending to 0.85 Å −1 and unilamellar vesicles for smaller q z. Modeling obtains the electron density profile and values for the area per molecule, for the locations of the component groups, and for the different types of thicknesses of the bilayer, such as the hydrocarbon thickness and the steric thickness.

The recently developed CMAP correction to the CHARMM22 force field (C22) is evaluated from 25 ns molecular dynamics simulations on hen lysozyme. Substantial deviations from experimental backbone root mean-square fluctuations and N-H NMR order parameters obtained in the C22 trajectories (especially in the loops) are eliminated by the CMAP correction. Thus, the C22/CMAP force field yields improved dynamical and structural properties of proteins in molecular dynamics simulations.

The traction forces developed by cells depend strongly on the substrate rigidity. In this letter, we characterize quantitatively this effect on MDCK epithelial cells by using a microfabricated force sensor consisting in a high-density array of soft pillars whose stiffness can be tailored by changing their height and radius to obtain a rigidity range from 2 nN/ μm up to 130 nN/ μm. We find that the forces exerted by the cells are proportional to the spring constant of the pillars meaning that, on average, the cells deform the pillars by the same amount whatever their rigidity. The relevant parameter may thus be a deformation rather than a force. These dynamic observations are correlated with the reinforcement of focal adhesions that increases with the substrate rigidity.