Project description:Understanding the molecular determinants underlying protein function requires the characterization of both structure and dynamics at atomic resolution. Nuclear relaxation rates allow a precise characterization of protein dynamics at the Larmor frequencies of spins. This usually limits the sampling of motions to a narrow range of frequencies corresponding to high magnetic fields. At lower fields one cannot achieve sufficient sensitivity and resolution in NMR. Here, we use a fast shuttle device where the polarization builds up and the signals are detected at high field, while longitudinal relaxation takes place at low fields 0.5 < B0 < 14.1 T. The sample is propelled over a distance up to 50 cm by a blowgun-like system in about 50 ms. The analysis of nitrogen-15 relaxation in the protein ubiquitin over such a wide range of magnetic fields offers unprecedented insights into molecular dynamics. Some key regions of the protein feature structural fluctuations on nanosecond time scales, which have so far been overlooked in high-field relaxation studies. Nanosecond motions in proteins may have been underestimated by traditional high-field approaches, and slower supra-?(c) motions that have no effect on relaxation may have been overestimated. High-resolution relaxometry thus opens the way to a quantitative characterization of nanosecond motions in proteins.
Project description:In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a "caging" strategy to trap the ?-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20?ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60??s, derived from a structural rearrangement in ?-sheet refolding. The event lasts 10 times longer than the timescale of ?-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a ?-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a ?-sheet.
Project description:To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 ?s. The instrument employs a micro-mixer with a mixing time of 2.7 ?s integrated with a 30 mm long flow-cell of 109 ?m optical path length constructed from two parallel sheets of silver foil; it produces ultraviolet-visible spectra that are linear in absorbance up to 3.5 with a spectral resolution of 0.4 nm. Each spectrum corresponds to a different reaction time determined by the distance from the mixer outlet, and by the fluid flow rate. The reaction progress is monitored in steps of 0.35 ?s for a total duration of ~600 ?s. As a proof of principle the instrument was used to study spontaneous protein refolding of pH-denatured cytochrome c. Three folding intermediates were determined: after a novel, extremely rapid initial phase with ? = 4.7 ?s, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 ?s and 345 ?s to a coordinatively saturated low-spin iron form in quasi steady state. The time-resolution specifications of our spectrometer for the first time open up the general possibility for comparison of real data and molecular dynamics calculations of biomacromolecules on overlapping time scales.
Project description:Aromatic organic deep-blue emitters that exhibit thermally activated delayed fluorescence (TADF) can harvest all excitons in electrically generated singlets and triplets as light emission. However, blue TADF emitters generally have long exciton lifetimes, leading to severe efficiency decrease, i.e., rolloff, at high current density and luminance by exciton annihilations in organic light-emitting diodes (OLEDs). Here, we report a deep-blue TADF emitter employing simple molecular design, in which an activation energy as well as spin-orbit coupling between excited states with different spin multiplicities, were simultaneously controlled. An extremely fast exciton lifetime of 750 ns was realized in a donor-acceptor-type molecular structure without heavy metal elements. An OLED utilizing this TADF emitter displayed deep-blue electroluminescence (EL) with CIE chromaticity coordinates of (0.14, 0.18) and a high maximum EL quantum efficiency of 20.7%. Further, the high maximum efficiency were retained to be 20.2% and 17.4% even at high luminance.
Project description:The oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. Increased flexibility has been observed for two regions: the loop between ?-helices ?2 and ?3 and the C-terminal half of ?-helix ?3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural preorganization play an important role in the control of the oligomerization process.
Project description:A method is proposed for efficient laser modification of fused silica and sapphire by means of a burst of femtosecond pulses having time separation in the range 10-3000?ps. Modification enhancement with the pulse separation increase in the burst was observed on the tens picoseconds scale. It is proposed that accumulated transient tensile strain in the excitation region plays a crucial role in modification by a sub-nanosecond burst.
Project description:Single polypyrrole (PPy) nanowire-based microfluidic aptasensors were fabricated using a one-step electrochemical deposition method. The successful incorporation of the aptamers into the PPy nanowire was confirmed by fluorescence microscopy image. The microfluidic aptasensor showed responses to IgE protein solutions in the range from 0.01 nM to 100 nM, and demonstrated excellent specificity and sensitivity with faster response and rapid stabilization times (~20 s). At the lowest examined IgE concentration of 0.01 nM, the microfluidic aptasensor still exhibited ~0.32% change in the conductance. The functionality of this aptasensor was able to be regenerated using an acid treatment with no major change in sensitivity. In addition, the detection of cancer biomarker MUC1 was performed using another microfluidic aptasensor, which showed a very low detection limit of 2.66 nM MUC1 compared to commercially available MUC1 diagnosis assay (800 nM).
Project description:Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100's of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.
Project description:We developed an electric-field exposure microchannel system with 230-nm thin-layer gold electrodes and interfaced it with a single living cell imaging station and a 10-ns electric-pulse (10 nsEP) generator. This design allows us to image intracellular molecules and structures, membrane transport, and viability of single leukemic cells (HL60) while the cells are exposed to 10 nsEPs of 0-179 kV/cm, permitting the study of subcellular responses within a nanosecond regime. The electrodes confine a thin-layer section of the cells exposed to 10 nsEPs, offering unprecedented high spatial resolution (230 nm in the z-direction of imaging plane and electric field) for imaging intracellular molecules of single cells affected by 10 nsEPs. We found that nucleic acids, membrane transport rates, and viability of single cells depend on the number and electric-field-strength (E) of 10 nsEPs, showing the cumulative effect of 10 nsEPs on intracellular molecules and structures and suggesting the possibility of tuning them one-pulse-at-a-time. Using a lower E (51 kV/cm) of 10 nsEPs, we could manipulate nucleic acids of single living cells without disrupting their cellular membrane and viability. As E increases to 80, 124, and 179 kV/cm, membrane integrity and viability of cells exhibit higher dependence on the number of 10 nsEPs in a nonlinear fashion, showing that a critical E and pulse number are needed to surmount cellular transport barriers and membrane integrity.
Project description:Target detection paradigms have been widely applied in the study of human cognitive functions, particularly those associated with arousal, attention, stimulus processing and memory. In EEG recordings, the detection of task-relevant stimuli elicits the P300 component, a transient response with latency around 300 ms. The P300 response has been shown to be affected by the amount of mental effort and learning, as well as habituation. Furthermore, trial-by-trial variability of the P300 component has been associated with inter-stimulus interval, target-to-target interval or target probability; however, understanding the mechanisms underlying this variability is still an open question. In order to investigate whether it could be related to the distinct cortical networks in which coherent intrinsic activity is organized, and to understand the contribution of those networks to target detection processes, we carried out a simultaneous EEG-fMRI study, collecting data from 13 healthy subjects during a visual oddball task. We identified five large-scale networks, that largely overlap with the dorsal attention, the ventral attention, the core, the visual and the sensory-motor networks. Since the P300 component has been consistently associated with target detection, we concentrated on the first two brain networks, the time-course of which showed a modulation with the P300 response as detected in simultaneous EEG recordings. A trial-by-trial EEG-fMRI correlation approach revealed that they are involved in target detection with different functional roles: the ventral attention network, dedicated to revealing salient stimuli, was transiently activated by the occurrence of targets; the dorsal attention network, usually engaged during voluntary orienting, reflected sustained activity, possibly related to search for targets.