Structural basis of fluorescence fluctuation dynamics of green fluorescent proteins in acidic environments

Green fluorescent proteins (GFPs) have become powerful markers for numerous biological studies due to their robust fluorescence properties, site-specific labeling, pH sensitivity, and mutations for multiple-site labeling. Fluorescence correlation spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic GFP mutants takes place on a time scale of 45-300 ms, depending on pH, and have been attributed to external proton transfer. Here we present experimental evidence indicating that conformational change in the protein &beta-barrel is a determining step for the external protonation of GFP-S65T (at low pH) using time-resolved fluorescence and polarization anisotropy measurements. While the average anionic fluorescence lifetime of GFP-S65T is reduced by approximately 18% over a pH range of 3.6-10.0, the fluorescence polarization anisotropy decays mostly as a single exponential with a rotational time of phi = 17 +/- 1 ns, which indicates an intact beta-barrel with a hydrodynamic volume of 78 +/- 5 nm3. In contrast, the total fluorescence (525 +/- 50 nm) of the excited neutral state of S65T reveals a strong correlation between the fluorescence lifetime, structural conformation, and pH. The average fluorescence lifetime of the excited neutral state of S65T as a function of pH yields pKa approximately 5.9 in agreement with literature values using steady-state techniques. In contrast to the intact beta-barrel at high pH, the anisotropy of neutral S65T (at pH 相似文献   

Structural flexibility in hydrated proteins

The flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. Protein flexibility is also implicated in denaturation, allowing proteins to take up nonactive conformations that have free energies close to that of the native state. High-frequency dielectric measurement can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. In this work, 14 hydrated globular proteins are investigated using this method. Four relaxation processes are identified, one of which, with a relaxation time of 19 ns, can be correlated with the sum of the number densities of protein glycine and alanine residues. A second with a relaxation time of 2 ns shows a dependence on the number of threonine residues. It is concluded that the dipolar peptide groups of the protein backbone associated with these residues are responsible for these dielectric responses, with the lower frequency dispersion being due to backbone mobility in the hydrophobic environment of the protein core and the higher frequency response being associated with mobility on the more hydrophilic protein surface. The correlation of protein backbone flexibility with particular side chains indicates that these protein motions are under the direct control of the amino acid sequence of the protein.     

Fractional Brownian dynamics in proteins

Correlation functions describing relaxation processes in proteins and other complex molecular systems are known to exhibit a nonexponential decay. The simulation study presented here shows that fractional Brownian dynamics is a good model for the internal dynamics of a lysozyme molecule in solution. We show that both the dynamic structure factor and the associated memory function fit well the corresponding analytical functions calculated from the model. The numerical analysis is based on autoregressive modeling of time series.     

Imaging ultrafast dynamics of molecules with laser-induced electron diffraction

We introduce a laser-induced electron diffraction method (LIED) for imaging ultrafast dynamics of small molecules with femtosecond mid-infrared lasers. When molecules are placed in an intense laser field, both low- and high-energy photoelectrons are generated. According to quantitative rescattering (QRS) theory, high-energy electrons are produced by a rescattering process where electrons born at the early phase of the laser pulse are driven back to rescatter with the parent ion. From the high-energy electron momentum spectra, field-free elastic electron-ion scattering differential cross sections (DCS), or diffraction images, can be extracted. With mid-infrared lasers as the driving pulses, it is further shown that the DCS can be used to extract atomic positions in a molecule with sub-angstrom spatial resolution, in close analogy to the standard electron diffraction method. Since infrared lasers with pulse duration of a few to several tens of femtoseconds are already available, LIED can be used for imaging dynamics of molecules with sub-angstrom spatial and a few-femtosecond temporal resolution. The first experiment with LIED has shown that the bond length of oxygen molecules shortens by 0.1 ? in five femtoseconds after single ionization. The principle behind LIED and its future outlook as a tool for dynamic imaging of molecules are presented.     

Structural effects of fluorine substitution in proteins

The consequences of substitution of fluorine for the para hydrogen of a phenylalanine residue in ribonuclease-S were investigated by conformational energy calculations using the AMBER force field. Both the fluorine-containing protein and the corresponding nonfluorinated material were subjected to conformational adjustment through energy minimization and the minimum energy structures so defined were compared. Fluorine substitution leads to small alterations in many atomic positions in the protein, with adjustments at at sites more than 0.5 nm from the fluorine appearing to be somewhat larger than those within the immediate vicinity of the fluorine. Several atoms proximate to the fluorine atom were observed to move toward the fluorine while others in the same vicinity move away. The greater bulk of the fluorine atom and the strongly different electronic properties of fluorine compared to hydrogen thus appear to be insufficient to cause a consistent, unidirectional change in nearest-neighbor interactions upon introduction of a fluorine atom into a protein structure. Virtually all changes in atomic positions that are predicted by these calculations would be barely detectable by a crystallographic study with a resolution of 0.2 nm.     

Structural determination of a molten (Li-K)Cl mixture of the eutectic composition by x-ray diffraction and molecular dynamics simulation

X-ray diffraction was performed for a molten (Li-K)CI mixture of the cutectic composition at 668 K. The three-dimensional structure of the melt was determined with the aid of molecular dynamics simulation.     

Macromolecular dynamics and free volume in polymer melts

The temperature dependence of the excess free volume, derived from the quasi-lattice theory of Simba and Somcynsky, above the glass-transition temperature for the non-crystalline component of four semicrystalline polymers [polyoxymethylene, poly(ethyleneterephthalate), polyethylene, and nylon-661] is used to facilitate the interpretation of ¹H-n.m.r. data. This method gives values for the crystalline content that agree well with values obtained by other methods. The correlation between the excess free vlume of the quasi-lattice and the segmental mobility of the noncrystalline component above the glass-transition temperature is discussed.     

Photoelectron dynamics of the cooper minimum in free molecules

The general properties of the Cooper minimum in molecules are reviewed. Experimental results from synchrotron radiation together with theoretical calculations are presented on both the partial cross sections and angular distribution parameters, β. A detailed examination of HCl is used as an example. Previously unpublished results on CCl _n H_4?n , CCl _n F_4?n , ethylene dichloride, I₂ and ICl are included. The Cooper minimum is largely discussed as a perturbation from atomic behavior, and is examined as a function of atomic number. The Cooper minimum is also examined as a function of chemical environment. Finally, needs for future research are briefly described.     

Structural studies of magnesium nitride fluorides by powder neutron diffraction

Samples of ternary nitride fluorides, Mg₃NF₃ and Mg₂NF have been prepared by solid state reaction of Mg₃N₂ and MgF₂ at 1323–1423 K and investigated by powder X-ray and powder neutron diffraction techniques. Mg₃NF₃ is cubic (space group: Pm3m) and has a structure related to rock-salt MgO, but with one cation site vacant. Mg₂NF is tetragonal (space group: I4₁/amd) and has an anti-LiFeO₂ related structure. Both compounds are essentially ionic and form structures in which nitride and fluoride anions are crystallographically ordered. The nitride fluorides show temperature independent paramagnetic behaviour between 5 and 300 K.     

The evolution dynamics of model proteins

Explicit simulations of protein evolution, where protein chains are described at a molecular, although simplified, level provide important information to understand the similarities found to exist between known proteins. The results of such simulations suggest that a number of evolutionary-related quantities, such as the distribution of sequence similarity for structurally similar proteins, are controlled by evolutionary kinetics and do not reflect an equilibrium state. An important result for phylogeny is that a subset of the residues of each protein evolve on a much larger time scale than the other residues.     

Structural correlations and cooperative dynamics in supercooled liquids

The relationships between diffusivity and the excess, pair and residual multiparticle contributions to the entropy are examined for Lennard-Jones liquids and binary glassformers, in the context of approximate inverse power law mappings of simple liquids. In the dense liquid where diffusivities are controlled by collisions and cage relaxations, Rosenfeld-type excess entropy scaling of diffusivities is found to hold for both crystallizing as well as vitrifying liquids. The crucial differences between the two categories of liquids emerge only when local cooperative effects in the dynamics result in significant caging effects in the time-dependent behaviour of the single-particle mean square displacement. In the case of glassformers, onset of such local cooperativity coincides with onset of deviations from Rosenfeld-type excess entropy scaling of diffusivities and increasing spatiotemporal heterogeneity. In contrast, for two- and three-dimensional liquids with a propensity to crystallise, the onset of local cooperative dynamics is sufficient to trigger crystallization provided that the liquid is sufficiently supercooled that the free energy barrier to nucleation of the solid phase is negligible. The state points corresponding to onset of transient caging effects can be associated with typical values, within reasonable bounds, of the excess, pair, and residual multiparticle entropy as a consequence of the isomorph-invariant character of the excess entropy, diffusivity and related static and dynamic correlation functions.     

Probing polar solvation dynamics in proteins: a molecular dynamics simulation analysis

Measurements of time-resolved Stokes shifts on picosecond to nanosecond time scales have been used to probe the polar solvation dynamics of biological systems. Since it is difficult to decompose the measurements into protein and solvent contributions, computer simulations are useful to aid in understanding the details of the molecular behavior. Here we report the analysis of simulations of the electrostatic interactions of the rest of the protein and the solvent with 11 residues of the immunoglobulin binding domain B1 of protein G. It is shown that the polar solvation dynamics are position-dependent and highly heterogeneous. The contributions due to interactions with the protein and with the solvent are determined. The solvent contributions are found to vary from negligible after a few picoseconds to dominant on a scale of hundreds of picoseconds. The origin for the latter is found to involve coupled hydration and protein conformational dynamics. The resulting microscopic picture demonstrates that a wide range of possibilities have to be considered in the interpretation of time-resolved Stokes shift measurements.     

自由高斯波包在量子相空间中的动力学研究

在Tortes—Vega和Frederick（简称T-F）量子相空间的理论框架下,将波函数表示成相应的极化形式,给．出了量子相空间中的含时Schrodinger方程的另一种等价表示形式,并且利用这种方法数值求解了自由高斯波包在相空间中的演化行为,对得到的结果进行了讨论,以期用这种方法探索更为实际和复杂的体系在量子相空间中的演化行为。     

Structural and thermodynamic investigations on the aggregation and folding of acylphosphatase by molecular dynamics simulations and solvation free energy analysis

Protein engineering method to study the mutation effects on muscle acylphosphatase (AcP) has been actively applied to describe kinetics and thermodynamics associated with AcP aggregation as well as folding processes. Despite the extensive mutation experiments, the molecular origin and the structural motifs for aggregation and folding kinetics as well as thermodynamics of AcP have not been rationalized at the atomic resolution. To this end, we have investigated the mutation effects on the structures and thermodynamics for the aggregation and folding of AcP by using the combination of fully atomistic, explicit-water molecular dynamics simulations, and three-dimensional reference interaction site model theory. The results indicate that the A30G mutant with the fastest experimental aggregation rate displays considerably decreased α1-helical contents as well as disrupted hydrophobic core compared to the wild-type AcP. Increased solvation free energy as well as hydrophobicity upon A30G mutation is achieved due to the dehydration of hydrophilic side chains in the disrupted α1-helix region of A30G. In contrast, the Y91Q mutant with the slowest aggregation rate shows a non-native H-bonding network spanning the mutation site to hydrophobic core and α1-helix region, which rigidifies the native state protein conformation with the enhanced α1-helicity. Furthermore, Y91Q exhibits decreased solvation free energy and hydrophobicity compared to wild type due to more exposed and solvated hydrophilic side chains in the α1-region. On the other hand, the experimentally observed slower folding rates in both mutants are accompanied by decreased helicity in α2-helix upon mutation. We here provide the atomic-level structures and thermodynamic quantities of AcP mutants and rationalize the structural origin for the changes that occur upon introduction of those mutations along the AcP aggregation and folding processes.     

Structural study of perovskite-type fine particles by synchrotron radiation powder diffraction

The crystal structures of BaTiO₃ and PbTiO₃ fine particles have been investigated by powder diffraction using synchrotron radiation high energy X-rays. It is revealed that a BaTiO₃ fine particle essentially consists of tetragonal and cubic structure components at 300 K, whereas a PbTiO₃ fine particle consists of a tetragonal structure. Adopting a structure model for the BaTiO₃ particle that a cubic shell covers a tetragonal core, the thickness of cubic BaTiO₃ shell is estimated at almost constant irrespective of particle sizes. Successive phase transitions are detected in 100 nm particles of BaTiO₃ near the phase-transition temperatures of a bulk crystal. The changes in diffraction profiles are small, but they are apparent for a most up-to-date powder diffractometry. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structural and thermodynamic aspects of electron diffraction by lithium fluoride dimers

Ivanovo Chemical Engineering Institute. Translated from Zhurnal Strukturnoi Khimii, Vol. 32, No. 1, pp. 60–67, January–February, 1991.     

Modeling solvent effects in molecular dynamics simulations of proteins

A series of computer simulations has been carried out on bovine pancreatic trypsin inhibitor using various models to mimic the effects of explicit bulk solvent on the structure of the protein. The solvent properties included are the polarization of the solute by the polar bulk solvent and the restraining effect on the motional freedom of the solute due to frictional drag at the solvent–protein surface interface. The former has been included by using a distance–dependent dielectric permittivity to screen the electrostatic interactions, whereas the latter is simulated by adding a limited number of solvent molecules near the protein surface. To achieve the proper mobility of the water molecules, their motion was restrained by adding a harmonic restraining force. It was found that a very small force constant was sufficient to model the static and dynamical behavior of the fully solvated solute, but that it was necessary to include enough explicit waters to occupy the first solvation shell. © 1992 John Wiley & Sons, Inc.