Model studies on p-hydroxybenzoate hydroxylase. The catalytic role of Arg-214 and Tyr-201 in the hydroxylation step

A model C-(4a)-flavinhydroperoxide (FlHOOH) is described that contains the tricyclic isoalloxazine moiety, the C-4a-hydroperoxide functionality, and a beta-hydroxyethyl group to model the effect of the 2'-OH group of the ribityl side chain of native FADHOOH. The electronic structures of this reduced flavin (H(3)()Fl(red)()), its N1 anion (H(2)()Fl(red)()(-)()), oxidized flavin (HFl(ox)()), and FlHOOH have been fully optimized at the B3LYP/ 6-31+G(d,p) level of theory. This model C-4a-flavinhydroperoxide is used to describe the transition state for the key step in the paradigm aromatic hydroxylase, p-hydroxybenzoate hydroxylase (PHBH): the oxidation of p-hydroxybenzoate (p-OHB). The Tyrosine-201 residue in PHBH is modeled by phenol, and Arginine-214 is modeled by guanidine. Electrophilic aromatic substitution proceeds by an S(N)2-like attack of the aromatic sextet of p-OHB phenolate anion on the distal oxygen of FlHOOH 3. The transition structure for oxygen atom transfer is fully optimized [B3LYP/6-31+G(d,p)] and has a classical activation barrier of 24.9 kcal/mol. These data suggest that the role of the Tyr-201 is to orient the p-OHB substrate and to properly align it for the oxygen transfer step. Although the negatively charged phenolate oxygen does activate the C-3 carbon of p-OHB phenolate anion toward oxidation relative to ortho oxidation of the carboxylate anion, it appears that H-bonding the Tyr-201 residue to this phenolic oxygen stabilizes both the ground state (GS) and the transition state (TS) approximately equally and therefore plays only a minor role, if any, in lowering the activation barrier. Complexation of p-OHB with guanidine has only a modest effect upon the oxidation barriers. When the complex is in the form of a salt-bridge (10a), the barrier is only slightly reduced. When the TSs are placed in THF solvent (COSMO) with full geometry optimization, salt-bridge TS-A is slightly favored (DeltaDeltaE() = 2.3 kcal/mol).     

Analysis of single-molecule dynamics in liquid HF

The center-of-mass velocity autocorrelation function of liquid HF is examined by means of a velocity field approach, originally developed for monatomic liquids and subsequently tested for liquid water. The basic ingredients of the approach (the wavevector-dependent current correlations) are evaluated by computer simulations in a model HF system using a recently developed intermolecular potential. The theory is found to reproduce the relevant features of self-dynamics in HF. The main advantage of the approach is the decomposition of single-molecule motion into longitudinal and transverse contributions, which provides information clarifying the origin of the peculiarities of HF dynamics. A comparison with the results for H₂O is presented, with the aim of stressing the different behavior of these two hydrogen-bonded systems.     

Toward accurate barriers for enzymatic reactions: QM/MM case study on p-hydroxybenzoate hydroxylase

The hydroxylation reaction catalyzed by p-hydroxybenzoate hydroxylase has been investigated by quantum mechanical/molecular mechanical (QM/MM) calculations at different levels of QM theory. The solvated enzyme was modeled (approximately 23,000 atoms in total, 49 QM atoms). The geometries of reactant and transition state were optimized for ten representative pathways using semiempirical (AM1) and density functional (B3LYP) methods as QM components. Single-point calculations at B3LYP/MM optimized geometries were performed with local correlation methods [LMP2, LCCSD(T0)] and augmented triple-zeta basis sets. A careful validation of the latter approach with regard to all computational parameters indicates convergence of the QM contribution to the computed barriers to within approximately 1 kcal mol(-1). Comparison with the available experimental data supports this assessment.     

Probing the charge-transfer dynamics in DNA at the single-molecule level

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.     

Conformational dynamics in nitrogen-fused azabicycles

Using molecular mechanics (MM3 force field)-based methodology, conformational dynamics have been studied for 1-azabicyclo[2.2.0]hexane, 1-azabicylo[3.3.0]octane, and 1-azabicylo[4.4.0]decane. Obtained conformational schemes describe the flexibity of these parent azabicyles as well as permit us to estimate conformational mobility in related N-fused systems. Quantum mechanics ab initio calculations have been used in order to check the reliability of molecular mechanics-provided estimates of relative energy of conformers. The previous dynamic NMR (DNMR) data have been reinterpreted for some polycyclic alkaloids.     

Conformational studies of haloperidol

The high field¹H,¹⁹F, and¹³C NMR spectra of haloperidol (HP) in CDCl₃ solution were recorded and analyzed with the aid of both homonuclear (H, H-COSY, H, H-COSYLR) and heteronuclear (H,C-COSY) chemical shift correlation experiments. Through space interactions between (1) the piperidine ring and the chlorinated phenyl ring and (2) the fluorinated phenyl ring and a methylene group were identified using phase sensitive Nuclear Overhauser and Exchange Spectroscopy (NOESY). The NOESY results are consistent with a planar structure rather than a folded version of haloperidol.     

Identifying mechanisms of interfacial dynamics using single-molecule tracking

The "soft" (i.e., noncovalent) interactions between molecules and surfaces are complex and highly varied (e.g., hydrophobic, hydrogen bonding, and ionic), often leading to heterogeneous interfacial behavior. Heterogeneity can arise either from the spatial variation of the surface/interface itself or from molecular configurations (i.e., conformation, orientation, aggregation state, etc.). By observing the adsorption, diffusion, and desorption of individual fluorescent molecules, single-molecule tracking can characterize these types of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, the fluorescence intensity or emission wavelength (in resonance energy transfer experiments) can be used to track the molecular configuration and simultaneously directly relate this to the resulting interfacial mobility or affinity. In this feature article, we review recent advances involving the use of single-molecule tracking to characterize heterogeneous molecule-surface interactions including multiple modes of diffusion and desorption associated with both internal and external molecular configuration, Arrhenius-activated interfacial transport, spatially dependent interactions, and many more.     

Revealing time bunching effect in single-molecule enzyme conformational dynamics

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.     

Ab initio molecular orbital studies of flavin radicals and the lowest triplet state of isoalloxazine

Ab initio calculations have been performed on the anion, neutral and cation radicals of isoalloxazine (isoalloxazine^?, 5H-isoalloxazine and 1,5-dihydroisoalloxazinium respectively) and on the lowest excited triplet state of isoalloxazine within the RHF and UHF SCF method. The calculated spin distribution is in reasonable agreement with experiments, provided the molecules are assumed to be planar. However, total energy considerations suggest that the radicals are non-planar in the gaseous phase.     

Conformational dynamics of a bispyridinium cyclophane

A complete study of the conformational behavior of 4,8-diaza-3(1,4),9(4,1)-dipyridina-1,6(1,4)-dibenzenacyclodecaphan-3(1),9(1)-bis(ilium) bishexafluorophosphate is described. This study allows us to conclude that the process observed by which the different chemical shifts of the pyridinium protons show coalescence at a high-temperature 1H NMR is the rotation around the C-N bond, whereas the conformational equilibrium between the four conformers is produced at low temperature.     

Studies in the alloxazine and isoalloxazine series

Alloxazine and lumichrome have been synthesized from symmetrical o-aminoazo compounds and barbituric acid. The yield of these compounds is considerably lower than when they are synthesized from unsymmetrical o-aminoazobenzenes and barbituric acid.For part XVII, see [1].     

The chemical dynamics of nanosensors capable of single-molecule detection

Recent advances in nanotechnology have produced the first sensor transducers capable of resolving the adsorption and desorption of single molecules. Examples include near infrared fluorescent single-walled carbon nanotubes that report single-molecule binding via stochastic quenching. A central question for the theory of such sensors is how to analyze stochastic adsorption events and extract the local concentration or flux of the analyte near the sensor. In this work, we compare algorithms of varying complexity for accomplishing this by first constructing a kinetic Monte Carlo model of molecular binding and unbinding to the sensor substrate and simulating the dynamics over wide ranges of forward and reverse rate constants. Methods involving single-site probability calculations, first and second moment analysis, and birth-and-death population modeling are compared for their accuracy in reconstructing model parameters in the presence and absence of noise over a large dynamic range. Overall, birth-and-death population modeling was the most robust in recovering the forward rate constants, with the first and second order moment analysis very efficient when the forward rate is large (>10(-3) s(-1)). The precision decreases with increasing noise, which we show masks the existence of underlying states. Precision is also diminished with very large forward rate constants, since the sensor surface quickly and persistently saturates.     

Conformational dynamics of the nicotinic acetylcholine receptor channel: a 35-ns molecular dynamics simulation study

The nicotinic acetylcholine receptor (AChR) is the paradigm of ligand-gated ion channels, integral membrane proteins that mediate fast intercellular communication in response to neurotransmitters. A 35-ns molecular dynamics simulation has been performed to explore the conformational dynamics of the entire membrane-spanning region, including the ion channel pore of the AChR. In the simulation, the 20 transmembrane (TM) segments that comprise the whole TM domain of the receptor were inserted into a large dipalmitoylphosphatidylcholine (DPPC) bilayer. The dynamic behavior of individual TM segments and their corresponding AChR subunit helix bundles was examined in order to assess the contribution of each to the conformational transitions of the whole channel. Asymmetrical and asynchronous motions of the M1-M3 TM segments of each subunit were revealed. In addition, the outermost ring of five M4 TM helices was found to convey the effects exerted by the lipid molecules to the central channel domain. Remarkably, a closed-to-open conformational shift was found to occur in one of the channel ring positions in the time scale of the present simulations, the possible physiological significance of which is discussed.     

Conformational dynamics for chemical sensing: simplicity and diversity

Controllable switching between two stable conformations of inherently dynamic chemical architectures can be exploited as a potent signal transduction mechanism for chemical sensing. In this article are highlighted selected examples of this emerging class of shape-adaptive molecular sensors that are built with simplicity in their operating principles but with diversity in their structural designs.     

Study of derivatives of the isoalloxazine series

Five new isoalloxazine derivatives are synthesized by condensing o-diamines with alloxan. A method of synthesizing ring-substituted monoalkylphenylenediamines by reducing the corresponding o-nitro-N-alkylanilines with hydrazine hydrate in the presence of Raney nickel is developed.For Part I see [10].     

Segment dynamics in thin polystyrene films probed by single-molecule optics

Single fluorescent molecules (represented by spheres with a volume equal to the actual van der Waals volume of the molecule) has been embedded in a polystyrene matrix (left). Such molecules act as probes for the study of polymer nanoscale (segmental scale) dynamics in thin films deposited on a glass cover slide (right).     

Conformational transition pathway in the allosteric process of calcium-induced recoverin: molecular dynamics simulations

Recoverin is an important neuronal calcium sensor (NCS) protein, which have been implicated in a wide range of Ca(2+) signaling events in neurons and photoreceptors. To characterize the conformational transition of recoverin from the myristoyl sequestered state to the extrusion state, a series of conventional molecular dynamics (CMD) and targeted molecular dynamics (TMD) simulations were performed. The 36.8 ns long CMD and TMD simulations on recoverin revealed a reliably conformational transition pathway, which can be viewed as a sequential two-stage process. A very important mechanistic conclusion from the present TMD simulations is that the hydrophobic and hydrophilic interactions modulate the allostery cooperatively in the conformational transition pathway. In the first stage, three salt-bridges broken between Lys-84 and Gly-124, between Lys-5 and Glu-103 and between Gly-16 and Lys-97 are major components to destabilize the structure of state T and trigger the swivel of the N- and C-terminal domains. In the second stage, the rupture of H-bond Phe-56-O(...)H(O)-Thr-21 leads to the two helices of EF-1 apart from each other, destabilizing the hydrophobic interactions of the myristoyl group with its environment, whereas the making of H-bond Leu-108-O(...)H(O)-Ser-72 helps the interfacial domain maintain its structural integrity during the course of the myristoyl extrusion. The molecular dynamics simulations results are beneficial to understanding the mechanism of how and why NCS proteins make progress in the photo-signal transduction processes. Further experimental and theoretical studies are still very desirable.     

Conformational dynamics of tetraisopropylmethane and of tetracyclopropylmethane

Tetraisopropylmethane (1) exists in solution as a mixture of two types of conformers (D2d and S4 time-averaged symmetry) in the ratio 93:7 at -110 degrees C, interconverting with a barrier of 9.7 kcal mol-1. Molecular mechanics calculations and the multiplicity of NMR signals at low temperature allow the assignment of these conformations. The only conformation populated in tetracyclopropylmethane (2) is the same type as the minor conformation (S4 time-averaged symmetry) populated in 1. 13C NMR spectra at about -180 degrees C show that degenerate versions of this conformation interconvert with a barrier of 4.5 kcal mol-1. Molecular mechanics calculations that characterize the six possible conformational types for these molecules, and the most important interconversion pathways, are reported. Calculated and experimental barriers match satisfactorily well.