Hydrogen Bonding Dynamics during Protein Folding of Reduced Cytochrome c: Temperature and Denaturant Concentration Dependence

AbstractFolding dynamics of reduced cytochrome c triggered by the laser-induced reduction method is investigated from a viewpoint of the intermolecular interaction change. Change of the diffusion coefficient of cytochrome c during the refolding process is traced in the time domain from the unfolded value to the native value continuously at various denaturant (guanidine hydrochloride (GdnHCl)) concentrations and temperatures. In the temperature range of 288 K–308K and GdnHCl concentration range of 2.5M–4.25M, the diffusion change can be analyzed well by the two-state model consistently. It was found that the m‡-value and the activation energy of the transition state from the unfolded state for the hydrogen bonding network change are surprisingly similar to that for the local structural change around the heme group monitored by the fluorescence quenching experiment. This agreement suggests the existence of common or similar fundamental dynamics including water molecular movement to control the refolding dynamics. The nature of the transition state is discussed

Pressure dependence of diffusion coefficient and orientational relaxation time for acetonitrile and methanol in water: DRISM/mode-coupling study

We present results of theoretical description and numerical calculation of the dynamics of molecular liquids based on the Reference Interaction Site Model / Mode-Coupling Theory. They include the temperature-pressure(density) dependence of the translational diffusion coefficients and orientational relaxation times for acetonitrile and methanol in water at infinite dilution. Anomalous behavior, i.e. the increase in mobility with density, is observed for the orientational relaxation time of methanol, while acetonitrile does not show any deviations from the usual. This effect is in qualitative agreement with the recent data of MD simulation and with experimental measurements, which tells us that presented theory is a good candidate to explain such kind of anomalies from the microscopical point of view and with the connection to the structure of the molecules.Comment: 10 pages, 2 eps-figures, 3 table

Confocal microphotoluminescence of InGaN-based light-emitting diodes

Spatially resolved photoluminescence (PL) of InGaN/GaN/AlGaN-based quantum-well-structured light-emitting diodes (LEDs) with a yellow-green light (530 nm) and an amber light (600 nm) was measured by using confocal microscopy. Submicron-scale spatial inhomogeneities of both PL intensities and spectra were found in confocal micro-PL images. We also found clear correlations between PL intensities and peak wavelength for both LEDs. Such correlations for yellow-green and amber LEDs were different from the reported correlations for blue or green LEDs. This discrepancy should be due to different diffusion, localization, and recombination dynamics of electron-hole pairs generated in InGaN active layers, and should be a very important property for influencing the optical properties of LEDs. In order to explain the results, we proposed a possible carrier dynamics model based on the carrier localization and partial reduction of the quantum confinement Stark effect depending on an indium composition in InGaN active layers. By using this model, we also considered the origin of the reduction of the emission efficiencies with a longer emission wavelength of InGaN LEDs with high indium composition

Time-resolved detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system

An intrinsically disordered protein, α-synuclein (αSyn), binds to negatively charged phospholipid membranes and adopts an α-helical structure. This conformational change is also induced by interaction with sodium dodecyl sulfate (SDS), which is an anionic surfactant used in previous studies to mimic membrane binding. However, while the structure of the αSyn and SDS complex has been studied widely by various static measurements, the process of structural change from the denatured state to the folded state remains unclear. In this study, the interaction dynamics between αSyn and SDS micelles was investigated using time-resolved measurements with a micro-stopped-flow system, which has been recently developed. In particular, the time-resolved diffusion based on the transient grating technique in combination with a micro-stopped-flow system revealed the gradual change in diffusion triggered by the presence of SDS micelles. This change is induced not only by binding to SDS micelles, but also by an intramolecular conformational change. It was interesting to find that the diffusion coefficient decreased in an intermediate state and then increased to the final state in the binding reaction. We also carried out stopped-flow-kinetic measurements of circular dichroism and intramolecular fluorescence resonance energy transfer, and the D change was assigned to the formation of a compact structure derived from the helix bending on the micelle

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cyclic adenosine 3', 5'-monophosphate (cAMP) in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC (FL-PAC) and the BLUF domain with the α3-helix. DSCC of FL-PAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics

Time-resolved detection of light-induced conformational changes of heliorhodopsin

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 mu s and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 mu s). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process

High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor.

The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P

Electronic Spectra of Hydrogen-Bonded Indazole in a Supersonic Free Jet

Fluorescence properties of indazole complexes with various protic molecules in supersonic jets are investigated. Indazole forms hydrogen-bonded complexes with acetic acid, formic acid, water and ethanol in jets and the 0-0 bands are shifted by 256, 243, 274 and 201 cm\u27 to the red of the 0-0 band of free indazole, respectively. The dispersed fluorescence spectra of these complexes are all similar indicating similar complex structures. Different from the cases of indazole-acid complexes in solution, no spectroscopic evidence was obtained in jets for excited state tautomerization or protonation. On the basis of the analysis of the fluorescence excitation and emission spectra, it is suggested that indazole-acid complexes in jets are bonded by a single hydrogen bond