Origins of concentration dependence of waiting times for single-molecule fluorescence binding

Binary fluorescence time series obtained from single-molecule imaging experiments can be used to infer protein binding kinetics, in particular, association and dissociation rate constants from waiting time statistics of fluorescence intensity changes. In many cases, rate constants inferred from fluorescence time series exhibit nonintuitive dependence on ligand concentration. Here we examine several possible mechanistic and technical origins that may induce ligand dependence of rate constants. Using aggregated Markov models, we show under the condition of detailed balance that non-fluorescent bindings and missed events due to transient interactions, instead of conformation fluctuations, may underly the dependence of waiting times and thus apparent rate constants on ligand concentrations. In general, waiting times are rational functions of ligand concentration. The shape of concentration dependence is qualitatively affected by the number of binding sites in the single molecule and is quantitatively tuned by model parameters. We also show that ligand dependence can be caused by non-equilibrium conditions which result in violations of detailed balance and require an energy source. As to a different but significant mechanism, we examine the effect of ambient buffers that can substantially reduce the effective concentration of ligands that interact with the single molecules. To demonstrate the effects by these mechanisms, we applied our results to analyze the concentration dependence in a single-molecule experiment EGFR binding to fluorophore-labeled adaptor protein Grb2 by Morimatsu et al. (PNAS,104:18013,2007).Comment: 11 pages, 4 figures; J. Chem. Phys., 137, 201

Two-level Chebyshev filter based complementary subspace method: pushing the envelope of large-scale electronic structure calculations

We describe a novel iterative strategy for Kohn-Sham density functional theory calculations aimed at large systems (> 1000 electrons), applicable to metals and insulators alike. In lieu of explicit diagonalization of the Kohn-Sham Hamiltonian on every self-consistent field (SCF) iteration, we employ a two-level Chebyshev polynomial filter based complementary subspace strategy to: 1) compute a set of vectors that span the occupied subspace of the Hamiltonian; 2) reduce subspace diagonalization to just partially occupied states; and 3) obtain those states in an efficient, scalable manner via an inner Chebyshev-filter iteration. By reducing the necessary computation to just partially occupied states, and obtaining these through an inner Chebyshev iteration, our approach reduces the cost of large metallic calculations significantly, while eliminating subspace diagonalization for insulating systems altogether. We describe the implementation of the method within the framework of the Discontinuous Galerkin (DG) electronic structure method and show that this results in a computational scheme that can effectively tackle bulk and nano systems containing tens of thousands of electrons, with chemical accuracy, within a few minutes or less of wall clock time per SCF iteration on large-scale computing platforms. We anticipate that our method will be instrumental in pushing the envelope of large-scale ab initio molecular dynamics. As a demonstration of this, we simulate a bulk silicon system containing 8,000 atoms at finite temperature, and obtain an average SCF step wall time of 51 seconds on 34,560 processors; thus allowing us to carry out 1.0 ps of ab initio molecular dynamics in approximately 28 hours (of wall time).Comment: Resubmitted version (version 2

Measurements of the methane relaxation times for application to the infrared emission models of the upper atmospheres of outer planets and Titan

The 7.8 micrometer emission from the nu(sub 4) band of methane (CH4) is a regularly observed feature in the stratosphere of all the giant planets and Titan. On Jupiter, enhancements in this emission are associated with the infrared hot spots in the auroral zone. Attempts to model this phenomenon in particular, and to understand the role of methane in general, have been hampered in part by a lack of adequate laboratory measurements of the collisional relaxation times for the nu(sub 3) and nu(sub 4) levels over the appropriate temperature range. To provide this needed data, a series of laboratory experiments were initiated. In the experimental arrangement the nu(sub3) band of methane is pumped at 3.3 micrometers using a pulsed infrared source (Nd:YAG/dye laser system equipped with a wave-length extender). The radiative lifetime of the nu(sub 3) level (approximately 37 ms) is much shorter than the nu(sub 4) lifetime (approximately 390 ms); however, a rapid V-V energy transfer rate ensures that the nu(sub 4) level is substantially populated. The photoacoustic technique is used to acquire relaxation rate information. The experiments are performed using a low-temperature, low-pressure cell. Experimental apparatus and technique are described. In addition some of the experimental difficulties associated with making these measurements are discussed and some preliminary results are presented

Protein-based molecular contrast optical coherence tomography with phytochrome as the contrast agent

We report the use of phytochrome A (phyA), a plant protein that can reversibly switch between two states with different absorption maxima (at 660 and 730 nm), as a contrast agent for molecular contrast optical coherence tomography (MCOCT). Our MCOCT scheme builds up a difference image revealing the distribution of phyA within a target sample from pairs of consecutive OCT A-scans acquired at a probe wavelength of 750 nm, both with and without additional illumination of the target sample with 660-nm light. We demonstrate molecular imaging with this new MCOCT modality in a target sample containing a mixture of 0.2% Intralipid and 83 µM of phyA

Spectral triangulation molecular contrast optical coherence tomography with indocyanine green as the contrast agent

We report a new molecular contrast optical coherence tomography (MCOCT) implementation that profiles the contrast agent distribution in a sample by measuring the agent's spectral differential absorption. The method, spectra triangulation MCOCT, can effectively suppress contributions from spectrally dependent scatterings from the sample without a priori knowledge of the scattering properties. We demonstrate molecular imaging with this new MCOCT modality by mapping the distribution of indocyanine green, a FDA-approved infrared red dye, within a stage 54 Xenopus laevis

Micro-resonator soliton generated directly with a diode laser

An external-cavity diode laser is reported with ultralow noise, high power coupled to a fiber, and fast tunability. These characteristics enable the generation of an optical frequency comb in a silica micro-resonator with a single-soliton state. Neither an optical modulator nor an amplifier was used in the experiment. This demonstration greatly simplifies the soliton generation setup and represents a significant step forward to a fully integrated soliton comb system.Comment: 7 pages, 5 figure