Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores.

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore.

DPD Simulation of Protein Conformations: From α-Helices to β-Structures.

We suggest a coarse-grained model for DPD simulations of polypeptides in solutions. The model mimics hydrogen bonding that stabilizes α-helical and β-structures using dissociable Morse bonds between quasiparticles representing the peptide groups amenable to hydrogen bonding. We demonstrate the capabilities of the model by simulating transitions between coil-like, globular, α-helical, and β-hairpin configurations of model peptides, varying Morse potential parameters, the hydrophobicities of residue side chains, and pH, which determines the charges of residue side chains.

The distribution of DNA translocation times in solid-state nanopores.

This paper systematically investigates the effects of solution viscosity, applied voltage and DNA chain length on the distribution of DNA translocation times through 8 ± 2 nm diameter silicon nitride nanopores. Linear dsDNA translocation events were selected based on the magnitude of current blockage and accumulated into scatter plots of current blockage and event duration (translocation time). The translocation time distribution was fitted to the solution of a Smoluchowski-type equation for 1D biased diffusion to a sink.

Determining nanocapillary geometry from electrochemical impedance spectroscopy using a variable topology network circuit model.

Solid-state nanopores and nanocapillaries find increasing use in a variety of applications including DNA sequencing, synthetic nanopores, next-generation membranes for water purification, and other nanofluidic structures. This paper develops the use of electrochemical impedance spectroscopy to determine the geometry of nanocapillaries. A network equivalent circuit element is derived to include the effects of the capacitive double layer inside the nanocapillaries as well as the influence of varying nanocapillary radius.

In vitro formation of amyloid from alpha-synuclein is dominated by reactions at hydrophobic interfaces.

Most in vitro investigations of alpha-Synuclein (alphaSyn) aggregation and amyloidogenesis use agitation in the presence of air and/or Teflon to accelerate kinetics. The effect of the agitation is implicitly or explicitly attributed to mass transfer or fibril fragmentation. This paper evaluates these hypotheses by agitating alphaSyn under typical amyloidogenic conditions with controlled numbers of balls made of polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), and borosilicate glass with no headspace.

Role of small oligomers on the amyloidogenic aggregation free-energy landscape.

We combine atomic-force-microscopy particle-size-distribution measurements with earlier measurements on 1-anilino-8-naphthalene sulfonate, thioflavin T, and dynamic light scattering to develop a quantitative kinetic model for the aggregation of beta-lactoglobulin into amyloid. We directly compare our simulations to the population distributions provided by dynamic light scattering and atomic force microscopy. We combine species in the simulation according to structural type for comparison with fluorescence fingerprint results.

Single-molecule protein unfolding in solid state nanopores.

We use single silicon nitride nanopores to study folded, partially folded, and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of beta-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins.

Information-theoretical analysis of time-correlated single-photon counting measurements of single molecules.

Time-correlated single photon counting allows luminescence lifetime information to be determined on a single molecule level. This paper develops a formalism to allow information theory analysis of the ability of luminescence lifetime measurements to resolve states in a single molecule. It analyzes the information content of the photon stream and the fraction of that information that is relevant to the state determination problem.

Separation and analysis of dynamic Stokes shift with multiple fluorescence environments: coumarin 153 in bovine beta-lactoglobulin A.

We use time-dependent fluorescence Stokes shift (TDFSS) information to study the fluctuation rates of the lipocalin, beta-lactoglobulin A in the vicinity of an encapsulated coumarin 153 molecule. The system has three unique dielectric environments in which the fluorophore binds. We develop a method to decompose the static and dynamic contributions to the spectral heterogeneity.

Beta-lactoglobulin assembles into amyloid through sequential aggregated intermediates.

We have investigated the aggregation and amyloid fibril formation of bovine beta-lactoglobulin variant A, with a focus on the early stages of aggregation. We used noncovalent labeling with thioflavin T and 1-anilino-8-naphthalenesulfonate to follow the conformational changes occurring in beta-lactoglobulin during aggregation using time resolved luminescence. 1-Anilino-8-naphthalenesulfonate monitored the involvement of the hydrophobic core/calyx of beta-lactoglobulin in the aggregation process.

Global fitting without a global model: regularization based on the continuity of the evolution of parameter distributions.

We introduce a new approach to global data fitting based on a regularization condition that invokes continuity in the global data coordinate. Stabilization of the data fitting procedure comes from probabilistic constraint of the global solution to physically reasonable behavior rather than to specific models of the system behavior. This method is applicable to the fitting of many types of spectroscopic data including dynamic light scattering, time-correlated single-photon counting (TCSPC), and circular dichroism.

COCIS: Markov processes in single molecule fluorescence.

This article examines the current status of Markov processes in single molecule fluorescence. For molecular dynamics to be described by a Markov process, the Markov process must include all states involved in the dynamics and the FPT distributions out of those states must be describable by a simple exponential law. The observation of non-exponential first-passage time distributions or other evidence of non-Markovian dynamics is common in single molecule studies and offers an opportunity to expand the Markov model to include new dynamics or states that improve understanding of the system.

Protein free energy landscapes remodeled by ligand binding.

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three components at high glucose concentration.

Hidden Markov model analysis of multichromophore photobleaching.

The interpretation of single-molecule measurements is greatly complicated by the presence of multiple fluorescent labels. However, many molecular systems of interest consist of multiple interacting components. We investigate this issue using multiply labeled dextran polymers that we intentionally photobleach to the background on a single-molecule basis.

Information theoretical approach to single-molecule experimental design and interpretation.

We use Shannon's definition of information to develop a theory to predict a photon-counting-based single-molecule experiment's ability to measure the desired property. We treat three phenomena that are commonly measured on single molecules: spectral fluctuations of a solvatochromic dye; assignment of the azimuthal dipole angle; determination of a distance by fluorescence resonant energy transfer using Förster's theory. We consider the effect of background and other "imperfections" on the measurement through the decrease in information.

Direct Determination of Kinetic Rates from Single-Molecule Photon Arrival Trajectories Using Hidden Markov Models.

The measurement of fluorescence from single protein molecules has become an important new tool in the study of dynamic processes, allowing for the direct visualization of the motions experienced by individual proteins and macromolecular complexes. The data from such single-molecule experiments are in the form of photon trajectories, consisting of arrival times and wavelength information on individual photons. The analysis of photon trajectories can be difficult, particularly if the motions are occurring at rates comparable to the photon arrival rate or in the presence of noise.

Copper Fluoride Luminescence during UV Photofragmentation of Bis(1,1,1,5,5,5-hexafluoro-2,4-pentanedionato)copper(II) in the Gas Phase.

Gas phase 308 and 350-370 nm photolysis of bis(1,1,1,5,5,5-hexafluoro-2,4-pentanedionato)copper(II), Cu(hfac)(2), produces CuF as well as copper atoms and dimers. These metal-containing fragments, identified by luminescence spectroscopy, are studied under a variety of gas phase conditions ranging from 1 bar in a static chamber to 10(-4) mbar in a collision-free molecular beam. Copper atom and dimer luminescence is observed at the higher pressures, whereas at low pressures (total pressure no greater than the vapor pressure of the sample) exclusively CuF emission is observed.