Thermodynamical properties of reaction intermediates during apoplastocyanin folding in time domain

Two intermediates observed for the folding process of apoplastocyanin (apoPC) were investigated by using a photoinduced triggering system combined with the transient grating and transient lens methods. The thermodynamic quantities, enthalpy, heat capacity, partial volume, and thermal expansion volume changes during the protein folding reaction were measured in time domain for the first time. An interesting observation is the positive enthalpy changes during the folding process. This positive enthalpy change must be compensated by positive entropy changes, which could be originated from the dehydration effect of hydrophobic residues and/or the translational entropy gain of bulk water molecules. Observed negative heat capacity change was explained by the dehydration effect of hydrophilic residues and/or motional confinement of amino acid side chains and water molecules in apoPC. The signs of the volume change and thermal expansion volume were different for two processes and these changes were interpreted in terms of the different relative contributions of the hydration and the dehydration of the hydrophilic residues. These results indicated two-step hydrophobic collapses in the early stage of the apoPC folding, but the nature of the dynamics was different.     

Diffusion coefficients as a monitor of reaction kinetics of biological molecules

For revealing spectrally silent dynamics in chemical reactions, a new method, the time-dependent diffusion coefficient, is presented. Principles and typical examples of this method, in particular applications to biologically related reactions, are reviewed. The pulsed laser induced transient grating signal of the photo-decomposition reaction of caged ATP showed that the diffusion coefficient increases gradually with time reflecting the molecular size decrease by the dissociation. Hence, this rate should be a direct measurement of the photo-dissociation rate of ATP from the caged state. In an application to a protein folding reaction, the time-development of the diffusion coefficient was observed during the folding reaction. This time dependence was interpreted in terms of the intermolecular interaction change; i.e., conversion from the intermolecular hydrogen bonding to intramolecular one. It was found that the change of the hydrogen bonding network occurred by the two state manner in entire refolding process of cytochrome c. The unique feature of this time-dependent diffusion coefficient method is discussed.     

Conformational and Intermolecular Interaction Dynamics of Photolyase/Cryptochrome Proteins Monitored by the Time‐Resolved Diffusion Technique

Cryptochrome (CRY), a blue light sensor protein, possesses a similar domain structure to photolyase (PHR) that, upon absorption of light, repairs DNA damage. In this review, we compare the reaction dynamics of these systems by monitoring the reaction kinetics of conformational change and intermolecular interaction change based on time‐dependent diffusion coefficient measurements obtained by using the pulsed laser‐induced transient grating technique. Using this method, time‐dependent biomolecular interactions, such as transient dissociation reactions in solution, have been successfully detected in real time. Conformational change in (6‐4) PHR has not been detected after the photoexcitation by monitoring the diffusion coefficient. However, the repaired DNA dissociates from PHR with a time constant of 50 μs, which must relate to a minor conformational change. However, CRY exhibits a considerable diffusion change with a time constant of 400 ms, which indicates that the protein–solvent interaction is changed by the conformational change. The C‐terminal domain of CRY is shown to be responsible for this change.     

Conformational relaxation dynamics of a poly(N-isopropylacrylamide) aqueous solution measured using the laser temperature jump transient grating method

We observed phase transition and phase relaxation processes of a poly(N-isopropylacrylamide) (PNIPAM) aqueous solution using the heterodyne transient grating (HD-TG) method combined with the laser temperature jump technique. The sample temperature was instantaneously raised by about 1.0 K after irradiation of a pump pulse to crystal violet (CV) molecules for heating, and the phase transition was induced for the sample with an initial temperature just below the lower critical solution temperature (LCST); the following phase relaxation dynamics was observed. Turbidity relaxation was observed in both the turbidity and HD-TG responses, while another relaxation process was observed only in the HD-TG response, namely via the refractive index change. It is suggested that this response is due to formation of globule molecules or their assemblies since they would have nothing to do with turbidity change but would affect the refractive index, which is dependent on the molar volume of a chemical species. Furthermore, the grating spacing dependence of the HD-TG responses suggests that the response was caused by the counter propagating diffusion of the coil molecules as a reactant species and the globule molecules as a product species and the lifetime of the globule molecules ranged from 1.5 to 5 seconds. Thus, we conclude that the turbidity reflects the dynamics of aggregate conditions, not molecular conditions. The coil and globule sizes were estimated from the obtained diffusion coefficient. The sizes of the coil molecules did not change at the initial temperatures below the LCST but increased sharply as it approaches LCST. We propose that the coil-state molecules associate due to hydrophobic interaction when the initial temperature was higher than LCST minus 0.5 K and that the globule-state molecules generated from the coil-state molecules showed a similar trend in temperature. The phase transition was also induced by heating under a microscope, and the relaxation process was followed using the fluorescence peak shift of a fluorescent molecule-labeled PNIPAM. The result also supports the existence of a globule molecule or its assembly remains for several seconds in the phase relaxation.     

Time-dependent intermolecular interaction during protein reactions

A recently developed method to monitor reaction kinetics of intermolecular interaction is presented in this perspective. This method is based on time-dependent diffusion coefficient measurements using the pulsed laser induced transient grating technique. Using this method, time dependent biomolecular interactions, such as transient association and dissociation reactions in solution, have been successfully detected in real time. The principles and particular applications are described. In particular, unique features of this time-dependent diffusion coefficient method are emphasized by comparison with other techniques.     

Conformational dynamics of phototropin 2 LOV2 domain with the linker upon photoexcitation

Conformational dynamics of LOV2 domain of phototropin, a plant blue light photoreceptor, is studied by the pulsed laser induced transient grating (TG) technique. The TG signal of LOV2 without the linker part to the kinase domain exhibits the thermal grating signal due to the heat releasing from the excited state and a weak population grating by the adduct formation. The diffusion coefficients of the adduct product after forming the chemical bond between the chromophore and Cys residue are found to be slightly smaller than that of the reactant, which implies that the core shrinks slightly on the adduct formation. After that change, no significant conformational change was observed. On the other hand, the signal of LOV2 with the linker part to the kinase domain clearly shows very different diffusion coefficients between the original and the adduct species. The large difference indicates significant global conformational change of the protein moiety upon the adduct formation. More interestingly, the diffusion coefficient is found to be time-dependent in the observation time range. The dynamics representing the global conformational change is a clear indication of a spectral silent intermediate between the excited triplet state and the signaling product. From the temporal profile analysis of the signal, the rate of the conformational change is determined to be 2 ms.     

Effect of Hydrated Ionic Liquid on Photocycle and Dynamics of Photoactive Yellow Protein

The mechanism by which proteins are solvated in hydrated ionic liquids remains an open question. Herein, the photoexcitation dynamics of photoactive yellow protein dissolved in hydrated choline dihydrogen phosphate (Hy[ch][dhp]) were studied by transient absorption and transient grating spectroscopy. The photocyclic reaction of the protein in Hy[ch][dhp] was similar to that observed in the buffer solution, as confirmed by transient absorption spectroscopy. However, the structural change of the protein during the photocycle in Hy[ch][dhp] was found to be different from that observed in the buffer solution. The known change in the diffusion coefficient of the protein was apparently suppressed in high concentrations of [ch][dhp], plausibly due to stabilization of the secondary structure.     

Transient dimerization and conformational change of a BLUF protein: YcgF

The photochemical reaction dynamics of YcgF, a BLUF protein, were investigated by the pulsed laser-induced transient grating (TG) technique. The TG signal showed three reaction time constants: 2.7 micros, 13 micros, and 2 ms. The fastest was tentatively attributed to relaxation of the excited triplet state of the chromophore, flavin adenine dinucleotide (FAD), and the others represented conformational changes of the protein. The TG signal provided clear evidence that the diffusion coefficient (D) of the photoproduct (3.8x10(-11) m2 s-1) was significantly less than that of the reactant (8.3x10(-11) m2 s-1), with a time constant of 2 ms at a protein concentration of 700 microM. Interestingly, the rate constant increased in proportion to the concentration of the protein, indicating that protein dimerization was one of the main reactions occurring after photoexcitation. The significant reduction in D indicates that a conformational change leading to an increase in interactions with water molecules occurs upon formation of the signaling state. The 13 mus dynamics was attributed to the conformational change that induced transient dimerization. This conformational change might be an essential process for the creation of the signaling state. A detailed scheme for the photochemical reaction of YcgF is proposed.     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

Intermolecular interaction of myoglobin with water molecules along the pH denaturation curve

A method of diffusion coefficient (D) measurement for proteins based on the pulsed laser-induced transient grating method using a photosensitive cross-linker was applied to the characterization of the pH denaturation process of holo- and apo-myoglobin (Mb) from the viewpoint of protein-water interaction. It was found that the pH denaturation curve monitored by D agrees quite well with that determined by the circular dichroism intensity for holo-Mb. This fact indicates that the changes in intermolecular interaction and the alpha-helix content occur simultaneously during the unfolding process. However, the pH dependence of D for apo-Mb was different from that of alpha-helix content. This different behavior can be explained in terms of the different denaturation steps for the secondary structure and the hydrogen bonding network of the intermediate species around pH 4; i.e., this intermediate is partially unfolded, but the hydrogen bonding network is dominantly an intramolecular one. Taking previously reported properties of this species into account, we conclude that water molecules are trapped in the hydrophobic core of the apo-Mb pH 4 intermediate. This fact suggests that the kinetic intermediate state of the protein folding process is a swollen state without water molecular exchange with the bulk phase.     

Diffusion of platinum ions and platinum nanoparticles during photoreduction processes using the transient grating method

The photoreduction process of PtCl(6)2- to Pt nanoparticles in poly(N-vinyl-2-pyrrolidone) solutions upon UV light irradiation was investigated by monitoring the change in the diffusion coefficient (D). The D values of chemical species during UV irradiation was measured by the laser-induced transient grating (TG) method. The TG signal of the PtCl(6)2- solution before UV irradiation was composed of three kinds of contributions, the thermal grating, the species grating due to the creation of PtCl4(2-), and the species grating due to the depletions of PtCl6(2-). Upon UV irradiation of the solution, the species grating signal due to PtCl6(2-) diminished and then the TG signal of Pt nanoparticles gradually appeared. This result indicates that the gradual clustering of Pt0 atoms into Pt nanoparticles occurs after all PtCl(6)2- ions are photochemically reduced to PtCl(4)2- and subsequently transformed to Pt0 atoms with a short delay. With increasing time of the UV irradiation, the TG signal intensity increased, while D of the Pt nanoparticles did not change. This suggests that the number of Pt nanoparticles increases, but the size of the Pt nanoparticles with the polymer layer is unchanged, in the course of the UV irradiation.     

Geometrical aspects of self diffusion in liquid water

In the late 1950s Cohen and Turnbull proposed a model for the self diffusion process in high density fluids, which considers the diffusion process as driven by the free volume fluctuations. An explicit expression was derived for the diffusion coefficient on hard sphere systems, and the results were consistent with molecular dynamic simulations. However the model is not able to reproduce experimental or simulation results for associated fluids, particularly in liquid water, where there is a huge variation of the diffusion coefficient without a significant change in the density. This implies that other properties, besides the free volume, have to be considered in modelling the diffusion process. The model we propose here takes into account both the volume and the shape of the “cage” surrounding the molecules. The “cage” is defined through the Voronoi polyhedra which are calculated from molecular dynamics simulations. The resulting diffusion coefficients, which have been calculated making use only of structural properties, are in good agreement with those evaluated directly from the long time behaviour of the molecular mean square displacements during the molecular dynamics runs.     

Investigation of Intrachain Exciton Diffusion of MEH-PPV in Solution with Different Polarity

Femtosecond time-resolved transient grating technique was adopted to insight into the intra-chain exciton diffusion of MEH-PPV in solution with different polarity. Broadband white-light continuum was introduced as the probe to observe the transient absorption and the femtosecond time-resolved transient grating information simultaneously. The vibrational dephasing behaviors, single exciton relaxation, and population relaxation dynamics of MEH-PPV were systematically investigated. The result shows that the relaxation processes of the sample solution will be accelerated in the solvent with larger polarity.     

Differential permeation — Part I: A method for the study of solvent diffusion through membranes

We describe the differential permeation method for the study of the diffusion of solvents from a liquid (or liquid mixture) through flat or tubular membranes. This method consists of measuring the transient permeation rates through the membrane when one of its faces is suddenly put into contact with the liquid medium. The change in the transient rate with time is analyzed by numerical best fitting methods to determine the Fickian diffusion coefficient. A simplified equation is proposed for the fitting of the response of a tubular membrane. Deviations from the Fickian transport mechanism with concentration-independent diffusion coefficient can be evidenced and eventually analyzed by using other mechanistic models.     

Experimental investigation of the Soret effect in acetone/water and dimethylsulfoxide/water mixtures

The thermal diffusion behavior of acetone/water and dimethylsulfoxide(DMSO)/water mixtures has been experimentally investigated by a transient holographic grating technique named thermal diffusion forced Rayleigh scattering (TDFRS). For both systems a sign change of the Soret coefficient S(T) with varying water content has been predicted by simulations [C. Nieto Draghi et al., J. Chem. Phys. 122, 114503 (2005)]. The sign change of S(T) is confirmed by the experiment. Except for equimolar concentrations of acetone/water the agreement between the experimental and simulation data is reasonable.     

N,N'-linked oligoureas as foldamers: chain length requirements for helix formation in protic solvent investigated by circular dichroism, NMR spectroscopy, and molecular dynamics

N,N'-linked oligoureas with proteinogenic side chains are peptide backbone mimetics belonging to the gamma-peptide lineage. In pyridine, heptamer 4 adopts a stable helical fold reminiscent of the 2.6(14) helical structure proposed for gamma-peptide foldamers. In the present study, we have used a combination of CD and NMR spectroscopies to correlate far-UV chiroptical properties and conformational preferences of oligoureas as a function of chain length from tetramer to nonamer. Both the intensity of the CD spectra and NMR chemical shift differences between alphaCH2 diastereotopic protons experienced a marked increase for oligomers between four and seven residues. No major change in CD spectra occurred between seven and nine residues, thus suggesting that seven residues could be the minimum length required for stabilizing a dominant conformation. Unexpectedly, in-depth NMR conformational investigation of heptamer 4 in CD3OH revealed that the 2.5 helix probably coexists with partially (un)folded conformations and that Z-E urea isomerization occurs, to some degree, along the backbone. Removing unfavorable electrostatic interactions at the amino terminal end of 4 and adding one H-bond acceptor by acylation with alkyl isocyanate (4 --> 7) was found to reinforce the 2.5 helical population. The stability of the 2.5 helical fold in MeOH is further discussed in light of unrestrained molecular dynamics (MD) simulation. Taken together, these new data provide additional insight into the folding propensity of oligoureas in protic solvent and should be of practical value for the design of helical bioactive oligoureas.     

Modulation of structure and dynamics by disulfide bond formation in unfolded states

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.     

Kinetics of the grating formation in holographic polymer-dispersed liquid crystals: NMR measurement of diffusion coefficients

Polymer films with embedded liquid crystal inclusions (polymer-dispersed liquid crystals) are superb composites for addressable windows, flexible displays and optical storage. Their scattering behavior and electro-optic properties depend essentially on the shape and size of the liquid crystal inclusions, which are typically formed by phase separation from a multicomponent homogeneous mixture. Here, pulsed field gradient NMR is used to measure the self-diffusion coefficients of the liquid crystal and a photo-reactive monomer, which compose such a precursor mixture. The kinetics of holographic grating formation in this mixture can be predicted by inserting the NMR diffusion coefficient of the monomer and the polymerization rate in a reaction diffusion model. The ratio of diffusion rate over reaction rate is found to be in the limiting regime in which the kinetics of the grating formation is not sensitive to this parameter.     

Viscosity scaling for the glassy phase of protein folding

Although commendable progress has been made in the understanding of the physics of protein folding, a key unresolved issue is whether Kramers' diffusion model of chemical reactions is generally applicable to activated barrier crossing events during folding. To examine the solvent viscosity effect on the folding transition of native-like trapped intermediates, laser flash photolysis has been used to measure the microsecond folding kinetics of a natively folded state of CO-liganded ferrocytochrome c (M-state) in the 1-250 cP range of glycerol viscosity at pH 7.0, 20 degrees C. The single rate coefficient for the folding of the M-state to the native state of the protein (i.e., the M --> N folding process) decreases initially when the solvent viscosity is low (<10 cP), but saturates at higher viscosity, indicating that Kramers model is not general enough for scaling the viscosity dependence of post-transition folding involving glassy dynamics. Analysis based on the Grote-Hynes idea of time dependent friction in conjunction with defect diffusion dynamics can account for the observed non-Kramers scaling.