Separation of a set of model proteins was tested on a microchip electrophoresis analytical platform capable of sample injection by two different electrokinetic mechanisms. A range of separation modes-microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography, and nanoparticle-based sieving-was tested on glass and polydimethylsiloxane/glass microchips and with silica-nanoparticle colloidal arrays. The model proteins calmodulin (18 kiloDalton), bovine serum albumin (66 kDa), and concanavalin (106 kDa) were labeled with Alexa Fluor 647 for laser-induced fluorescence detection. The best separation and resolution were obtained in a silica-nanoparticle colloidal array chip.

The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in-vitro crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide through a process that involves transfer of electrons sequentially from FAD to FMN in the reductase domain of the enzyme and then from FMN to a heme in the oxygenase domain of the partner enzyme in a homodimeric complex. Efficient NO synthase activity is enabled by binding of the calcium signaling protein calmodulin (CaM) to a CaM-binding domain situated between the reductase and oxygenase domains. Because the rate of electron transfer depends sharply on distance, electron transfer requires close proximity of electron-transfer donor and acceptor. The sequence of electron transfers in NOS therefore necessitates multiple conformational states of the enzyme, suggesting that the electron transfers are conformationally gated. We have used fluorescence-labeled CaM and time-resolved fluorescence to detect the presence of multiple conformational states of NOS. Fluorescence is quenched by FRET to the heme groups of the enzyme, and the extent of quenching depends on the conformational state of the enzyme. We used site-directed mutants of both NOS and CaM to assign fluorescence quenching states to conformational states of the enzyme. Mutations were chosen that have known effects on enzyme kinetics and/or subdomain interactions. The results suggest the presence of multiple conformation states in which CaM is in close proximity to the oxygenase domain. We suggest that CaM docking to the oxygenase domain facilitates interaction of the FMN domain with the partner oxygenase domain. Such a conformation would enable electron transfer from FMN to the heme.

Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 3(10) helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 3(10) helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.

Observation of single molecules requires probing a sample volume that contains no more than one molecule at a time. For diffraction-limited probe volumes this requires molecular concentrations that may be incompatible with the biological system being interrogated. In this chapter we describe the use of nanofluidic devices as an alternative approach to restrict probe volumes below diffraction-limited values. In particular, we will focus on the use of nanoscale devices for the detection and optical mapping of single DNA molecules. The chapter describes unique electrokinetic phenomena that emerge on the nanoscale as well. The driving forces for fluid flow and interactions with the fluid via walls are qualitatively different on the nanoscale as compared to the microscale. Overlap of the electric double layers generated by opposite walls on the nanoscale alters the velocity profile (plug-like on the microscale to parabolic on the nanoscale for electrokinetic flows). The conformation of DNA is sensitive to channel dimensions. In nanochannels, DNA molecules stretch to nearly their full contour length when the channel dimensions are comparable to the persistence length of the DNA molecule. Methods of fabrication of nanochannel devices are surveyed. Electron beam lithography, focused ion beam milling, and nanoimprint lithography have been used to generate nanoscale devices. Finally, we describe recent applications of fluorescence imaging of single molecules in nanochannels for optical mapping of DNA and the detection of sequence variations.

To fully exploit the power of coherent Raman imaging, techniques are needed to image more than one vibrational frequency simultaneously. We describe a method for switching between two vibrational frequencies based on a single fiber-laser source. Stokes pulses were generated by soliton self-frequency shifting in a photonic crystal fiber. Pump and Stokes pulses were stretched to enhance vibrational resolution by spectral focusing. Stokes pulses were switched between two wavelengths on the millisecond time scale by a liquid-crystal retarder. Proof-of-principle is demonstrated by coherent anti-Stokes Raman imaging of polystyrene beads embedded in a poly(methyl methacrylate) (PMMA) matrix. The Stokes shift was switched between 3,050 cm(-1), where polystyrene has a Raman transition, and 2,950 cm(-1), where both polystyrene and PMMA have Raman resonances. The method can be extended to multiple vibrational modes.

Multi-domain oxidoreductases are a family of enzymes that catalyze oxidation-reduction reactions through a series of electron transfers. Efficient electron transfer requires a sequence of protein conformations that position electron donor and acceptor domains in close proximity to each other so that electron transfer can occur efficiently. An example is mammalian nitric oxide synthase (NOS), which consists of an N-terminal oxygenase domain containing heme and a C-terminal reductase domain containing NADPH/FAD and FMN subdomains. We describe the use of time-resolved and single-molecule fluorescence to detect and characterize the conformations and conformational dynamics of the neuronal and endothelial isoforms of NOS. Fluorescence signals are provided by a fluorescent dye attached to the Ca2+-signaling protein calmodulin (CaM), which regulates NOS activity. Time-resolved fluorescence decays reveal the presence of at least four underlying conformational states that are differentiated by the extent of fluorescence quenching. Single-molecule fluorescence displays transitions between conformational states on the time scales of milliseconds to seconds. This review describes the type of information available by analysis of time-resolved and single-molecule fluorescence experiments.

Nitric oxide synthase (NOS) generates a signaling molecule, nitric oxide, which is essential for processes such as vasodilation and neurotransmission. The enzyme forms homodimers containing cofactors FAD, FMN, and heme, each housed in subunits of the enzyme. Synthase activity in NOS is activated by calmodulin (CaM), which binds to a region adjacent to the FMN domain. Conformational motion is necessary to position sequential electron donor-acceptor pairs adjacent to one another for transfer of electrons from the FAD domain to the FMN domain and then to the oxygenase domain of the opposite member of the homodimer. We characterized the conformations and dynamics of endothelial and neuronal NOS with bound CaM by time-resolved and single-molecule detection of fluorescence from a fluorophore attached to CaM. Maximum entropy analysis of fluorescence decays for both neuronal and endothelial NOS reveals four conformational states, resolved by their distinct extents of quenching by energy transfer to heme. Clues to the identity of each population are found from their dependence on calcium concentration and from mutations known to disrupt specific interactions. The CaM mutation K115E, known to disrupt docking of CaM to the oxygenase domain, abolishes the two shortest lifetime populations. Single-molecule trajectories reveal conformational interchange on the millisecond to second time scales and permit analysis of the rates of interchange of conformational states. Exchange appears slowest to and from the conformation with highest quenching, consistent with the identification of this conformation as the "output" state of the enzyme.

We investigate the roles of measurement time scale and the nature of the fluorophores in the FRET states measured for calmodulin, a calcium signaling protein known to undergo pronounced conformational changes. The measured FRET distributions depend markedly on the measurement time scale (nanosecond or microsecond). Comparison of FRET distributions measured by donor fluorescence decay with FRET distributions recovered from single-molecule burst measurements binned over time scales of 90 μs to 1 ms reveals conformational averaging over the intervening time regimes. We find further that, particularly in the presence of saturating Ca2+, the nature of the measured single-molecule FRET distribution depends markedly on the identity of the FRET pair. The results suggest interchange between conformational states on time scales of hundreds of microseconds or less. Interaction with a fluorophore such as the dye Texas Red alters both the nature of the measured FRET distributions and the dynamics of conformational interchange. The results further suggest that the fluorophore may not be merely a benign reporter of protein conformations in FRET studies, but may in fact alter the conformational landscape.

Fluorescence anisotropy decay measurements and all atom molecular dynamics simulations are used to characterize the orientational motion and preferential interaction of a peptide, N-acetyl-tryptophan-amide (NATA) containing two peptide bonds, in aqueous, urea, guanidinium chloride (GdmCl), and proline solution. Anisotropy decay measurements as a function of temperature and concentration showed moderate slowing of reorientations in urea and GdmCl and very strong slowing in proline solution, relative to water. These effects deviate significantly from simple proportionality of peptide tumbling time to solvent viscosity, leading to the investigation of microscopic preferential interaction behavior through molecular dynamics simulations. Examination of the interactions of denaturants and osmolyte with the peptide backbone uncovers the presence of strongest interaction with urea, intermediate with proline, and weakest with GdmCl. In contrast, the strongest preferential solvation of the peptide side chain is by the nonpolar part of the proline zwitterion, followed by urea, and GdmCl. Interestingly, the local density of urea around the side chain is higher, but the GdmCl distribution is more organized. Thus, the computed preferential solvation of the side chain by the denaturants and osmolyte can account for the trend in reorientation rates. Analysis of water structure and its dynamics uncovered underlying differences between urea, GdmCl, and proline. Urea exerted the smallest perturbation of water behavior. GdmCl had a larger effect on water, slowing kinetics and stabilizing interactions. Proline had the largest overall interactions, exhibiting a strong stabilizing effect on both water-water and water-peptide hydrogen bonds. The results for this elementary peptide system demonstrate significant differences in microscopic behavior of the examined solvent environments. For the commonly used denaturants, urea tends to form disorganized local aggregates around the peptide groups and has little influence on water, while GdmCl only forms specific interactions with the side chain and tends to destabilize water structure. The protective osmolyte proline has the strongest and most specific interactions with the tryptophan side chain, and also stabilizes both water-water and water-peptide hydrogen bonds. Our results strongly suggest protein or peptide denaturation triggered by urea occurs by direct interaction, whereas GdmCl interacts favorably with side chains and destabilizes peptide-water hydrogen bonds. The stabilization of biopolymers by an osmolyte such as proline is governed by favorable preferential interaction with the side chains and stabilization of water.

The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of < 100 nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca2+. Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from < 0.01 μm2 s−1 to ~ 5 μm2 s−1, whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of > 10 μm2 s−1. For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells.

Activation of endothelial nitric oxide synthase (eNOS) by calmodulin (CaM) facilitates formation of a sequence of conformational states that is not well understood. Fluorescence decays of fluorescently labeled CaM bound to eNOS reveal four distinct conformational states and single-molecule fluorescence trajectories show multiple fluorescence states with transitions between states occurring on time scales of milliseconds to seconds. A model is proposed relating fluorescence quenching states to enzyme conformations. Specifically, we propose that the most highly quenched state corresponds to CaM docked to an oxygenase domain of the enzyme. In single-molecule trajectories, this state occurs with time lags consistent with the oxygenase activity of the enzyme.

Kinetics of Interactions between LOV domains from Chlamydomonas reinhardtii Carey K. Johnson, Kathrin Magerl, Katee Wyant, Ashley McDade, Will Newhart, David C. Arnett, and Bernhard Dick. We have investigated the kinetics of interactions between Light-Oxygen-Voltage (LOV) domains of the light-sensing protein phototropin from Chlamydomonas reinhardtii. The photochemical response of phototropin to blue light involves adduct formation between a flavin mononucleotide cofactor and a nearby Cys residue. Interactions between LOV domains, either domains LOV1 and LOV2 within the same phototropin, or between LOV domains from different phototropin molecules, are thought to play a role in the subsequent activation of the phototropin kinase domain. We studied the kinetics of exchange of LOV domains between complexes (dimers or higher order oligomers) by stop-flow FRET measurements. A biphasic response with time constants from tens to hundreds of seconds was sensitive to prior exposure of LOV domains to blue light. LOV-LOV interactions have also been detected at the single-molecule level by TIRF imaging, providing further information about the interaction kinetics.

We demonstrate multi-mode microscopy based on a single femtosecond fiber laser. Coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS) and photothermal images can be obtained simultaneously with this simplified setup. Distributions of lipid and hemoglobin in sliced mouse brain samples and blood cells are imaged. The dependency of signal amplitude on the pump power and pump modulation frequency is characterized, which allows to isolate the impact from different contributions.

We present a combined experimental and computational study of unfolding pathways of a model 21-residue α-helical heteropeptide (W1H5-21) and a 16-residue β-hairpin (GB41-56). Experimentally, we measured fluorescence energy transfer efficiency as a function of temperature, employing natural tryptophans as donors and dansylated lysines as acceptors. Secondary structural analysis was performed with circular dichroism and Fourier transform infrared spectroscopy. Our studies present markedly different unfolding pathways of the two elementary secondary structural elements. During thermal denaturation, the helical peptide exhibits an initial decrease in length, followed by an increase, while the hairpin undergoes a systematic increase in length. In the complementary computational part of the project, we performed microsecond length replica-exchange molecular dynamics simulations of the peptides in explicit solvent, yielding a detailed microscopic picture of the unfolding processes. For the α-helical peptide, we found a large heterogeneous population of intermediates that are primarily frayed single helices or helix-turn-helix motifs. Unfolding starts at the termini and proceeds through a stable helical region in the interior of the peptide but shifted off-center toward the C-terminus. The simulations explain the experimentally observed non-monotonic variation of helix length with temperature as due primarily to the presence of frayed-end single-helix intermediate structures. For the β-hairpin peptide, our simulations indicate that folding is initiated at the turn, followed by formation of the hairpin in zipper-like fashion, with Cα···Cα contacts propagating from the turn to termini and hairpin hydrogen bonds forming in parallel with these contacts. In the early stages of hairpin formation, the hydrophobic side-chain contacts are only partly populated. Intermediate structures with low numbers of β-hairpin hydrogen bonds have very low populations. This is in accord with the "broken zipper" model of Scheraga. The monotonic increase in length with temperature may be explained by the zipper-like breaking of the hairpin hydrogen bonds and backbone contacts.