Effect Of Protein Environment Research Articles

Catalytic Mechanism and Energy Barriers for Butyrylcholinesterase-Catalyzed Hydrolysis of Cocaine

The geometries of the transition states, intermediates, and prereactive enzyme-substrate complex and the corresponding energy barriers have been determined by performing hybrid quantum mechanical/molecular mechanical (QM/MM) calculations on butyrylcholinesterase (BChE)-catalyzed hydrolysis of (−)- and (+)-cocaine. The energy barriers were evaluated by performing QM/MM calculations with the QM method at the MP2/6-31+G* level and the MM method using the AMBER force field. These calculations allow us to account for the protein environmental effects on the transition states and energy barriers of these enzymatic reactions, showing remarkable effects of the protein environment on intermolecular hydrogen bonding (with an oxyanion hole), which is crucial for the transition state stabilization and, therefore, on the energy barriers. The calculated energy barriers are consistent with available experimental kinetic data. The highest barrier calculated for BChE-catalyzed hydrolysis of (−)- and (+)-cocaine is associated with the third reaction step, but the energy barrier calculated for the first step is close to the highest and is so sensitive to the protein environment that the first reaction step can be rate determining for (−)-cocaine hydrolysis catalyzed by a BChE mutant. The computational results provide valuable insights into future design of BChE mutants with a higher catalytic activity for (−)-cocaine.

Modeling Effects of Oxyanion Hole on the Ester Hydrolysis Catalyzed by Human Cholinesterases

Molecular dynamics (MD) simulations and hydrogen bonding energy (HBE) calculations have been performed on the prereactive enzyme-substrate complexes (ES), transition states (TS1), and intermediates (INT1) for acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine (ACh), butyrylcholinesterase (BChE)-catalyzed hydrolysis of ACh, and BChE-catalyzed hydrolysis of (+)/(-)-cocaine to examine the protein environmental effects on the catalytic reactions. The hydrogen bonding of cocaine with the oxyanion hole of BChE is found to be remarkably different from that of ACh with AChE/BChE. Whereas G121/G116, G122/G117, and A204/A199 of AChE/BChE all can form hydrogen bonds with ACh to stabilize the transition state during the ACh hydrolysis, BChE only uses G117 and A199 to form hydrogen bonds with cocaine. The change of the estimated total HBE from ES to TS1 is ca. -5.4/-4.4 kcal/mol for AChE/BChE-catalyzed hydrolysis of ACh and ca. -1.7/-0.8 kcal/mol for BChE-catalyzed hydrolysis of (+)/(-)-cocaine. The remarkable difference of approximately 3 to 5 kcal/mol reveals that the oxyanion hole of AChE/BChE can lower the energy barrier of the ACh hydrolysis significantly more than that of BChE for the cocaine hydrolysis. These results help to understand why the catalytic activity of AChE against ACh is considerably higher than that of BChE against cocaine and provides valuable clues on how to improve the catalytic activity of BChE against cocaine.

Spectroscopic Properties of the Carotenoid 3‘-Hydroxyechinenone in the Orange Carotenoid Protein from the CyanobacteriumArthrospira maxima

The cyanobacterial water-soluble orange carotenoid binding protein (OCP) is an ideal system for study of the effects of protein environment on photophysical properties of carotenoids. It contains a single pigment, the carotenoid 3'-hydoxyechinenone (hECN). In this study, we focus on spectroscopic properties of hECN in solution and in the OCP, aiming to elucidate the spectroscopic effects of the carotenoid-protein interaction in the context of the function(s) of the OCP. The noncovalent binding of hECN to the OCP causes a conformational change in the hECN, leading to a prolongation of the effective conjugation length. This change is responsible for shortening of the S(1) lifetime from 6.5 ps in solution to 3.3 ps in the OCP. The conformational change and the hydrogen bonding via the carbonyl group of hECN result in stabilization of an intramolecular charge-transfer (ICT) state. No signs of the ICT state were found in hECN in solution, regardless of the solvent polarity; spectral bands in transient absorption spectra of OCP-bound hECN exhibit features typical for the ICT state. Application of global fitting analysis revealed further effects of binding hECN in the OCP. The S(1) state of hECN in the OCP decays with two time constants of 0.9 and 3.3 ps. Modeling of the excited-state processes suggests that these two components are due to two populations of hECN in the OCP that differ in the hydrogen bonding via the carbonyl group. These results support the hypothesis that the OCP functions as a photoprotective shield under excess light. Mechanistically, the broadening of the hECN absorption spectrum upon binding to OCP enhances filtering effect of hECN. Furthermore, the binding-induced conformational change and activation of the ICT state that leads to a shortening of hECN lifetime effectively makes the protein-bound hECN a more effective energy dissipator.

First-principles studies of C-13 NMR chemical shift tensors of amino acids in crystal state

A series of gauge-independent atomic orbital (GIAO) magnetic shielding calculations with ab initio Hartree–Fock (HF) and density-functional theory (DFT) methods were performed on alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, serine, and tyrosine in crystal state to study their 13C NMR chemical shift tensors. The GIAO calculations on the amino acids were performed with and without inclusion of the crystal environment. Compared to previous computational studies reported in literature, this study accounts for more complete crystal environmental effects on the 13C NMR chemical shifts and, therefore, provides calculated results in better agreement with the experimental data. For each amino acid considered, the crystal environment included in the calculations includes all molecules having hydrogen bonds with the target molecule. It has been shown that the 13C NMR chemical shift tensors, particularly the principal components δ 11 and δ 22, calculated without inclusion of the crystal environment significantly differ from the corresponding experimental data for the carbon atoms that are close to a hydrogen bond in the crystal, although the calculated isotropic 13C chemical shifts are in good agreement with the experimental data. The GIAO calculations including the crystal environment with both the HF and DFT methods can significantly improve the calculated 13C chemical shift tensors. The results calculated with the DFT method are slightly better than the corresponding results with the HF method. The good agreement between the calculated and experimental results indicate that the significant crystal environmental effects on the principal components of 13C chemical shift tensors of the amino acids are mainly attributed to the hydrogen bonding of amino acid with its environment. When all the hydrogen bonds of amino acid with its environment are included explicitly in the GIAO calculation with a supermolecule model, the calculated 13C chemical shift tensors are in good agreement with the corresponding experimental data. This insight obtained from studying the crystal environmental effects on 13C chemical shift tensors of amino acids in crystal state could also be useful for future computational studies of the protein environmental effects on 13C chemical shift tensors of amino acid residues of proteins.

Fluorophore Conformation in Green Fluorescent Protein: A Quantum Mechanics/Molecular Mechanics Study

AbstractGreen Fluorescent Protein (GFP) is a widely used fluorescent marker exhibiting two excitation peaks – a strong peak at 398 nm and a second at 475 nm, with the fluorescence at ca. 510 nm. Its molecular structure consists of a β-barrel composed of 11 β-strands and a central helix containing the fluorophore. Two different forms of the fluorophore – a protonated/neutral fluorophore and a de-protonated/anionic fluorophore – are thought to be responsible for the two distinct spectroscopic states. Notably, the isolated fluorophore in solution is efficiently quenched. Conformational flexibility within the protein cavity is an implicitly important factor that governs the photochemistry of GFP. However, the literature contains accounts of studies that reach conflicting conclusions, claiming that either the fluorophore's barrier to internal rotation is negligibly small or that the protein cavity is not complementary to a planar fluorophore. In this work, we calculate the torsional potential of one of the two exocyclic bonds that connect the two rings in the fluorophore, taking into account its immediate environment by applying a quantum mechanics/molecular mechanics method, with the ultimate aim of evaluating the protein-environment effects on the fluorescence.

Effect of Protein Environment on pKa Shifts in the Active Site of Photoactive Yellow Protein

During the photocycle of photoactive yellow protein (PYP), the pKa values of the chromophore (S-methyl p-coumarate) and Glu46 undergo drastic changes. In the dark state (pG), the chromophore is deprotonated and Glu46 is protonated. In the blue-shifted intermediate called pB, their protonation states are inverted. These pKa changes are thought to be closely related to the mechanism of signal transduction leading to bacterial phototaxis. In this study, we investigate the physical origin of the above pKa shifts using quantum chemical calculations at the level of ab initio Hartree−Fock (HF), semiempirical AM1, and density functional theory (DFT). Here, S-methyl p-coumarate and acetic acid (propionic acid) are chosen as models of the chromophore and Glu46, respectively. First, the protein environment surrounding these residues is approximated by a dielectric continuum. The HF and DFT calculations coupled with a continuum solvent model (SCIPCM or COSMO) reveal that the relative pKa (ΔpKa) of acetic acid to the ...

Short-strong hydrogen bonds and a low barrier transition state for the proton transfer reaction in RNase A catalysis: a quantum chemical study

There is growing evidence that some enzymes catalyze reactions through the formation of short–strong hydrogen bonds as first suggested by Gerlt and Gassman. Support comes from several experimental and quantum chemical studies that include correlation energies on model systems. In the present study, the process of proton transfer between hydroxyl and imidazole groups, a model of the crucial step in the hydrolysis of RNA by the enzymes of the RNase A family, is investigated at the quantum mechanical level of density functional theory and perturbation theory at the MP2 level. The model focuses on the nature of the formation of a complex between the important residues of the protein and the hydroxyl group of the substrate. We have also investigated different configurations of the ground state that are important in the proton transfer reaction. The nature of bonding between the catalytic unit of the enzyme and the substrate in the model is investigated by Bader's atoms in molecule theory. The contributions of solvation and vibrational energies corresponding to the reactant, the transition state and the product configurations are also evaluated. Furthermore, the effect of protein environment is investigated by considering the catalytic unit surrounded by complete proteins—RNase A and Angiogenin. The results, in general, indicate the formation of a short–strong hydrogen bond and the formation of a low barrier transition state for the proton transfer model of the enzyme.

Glutathione Transferase: A First-Principles Study of the Active Site

We present a first-principles study of the interaction of glutathione (GSH) with the enzyme glutathione transferase (GST) and its Y6F mutant. By comparing a reduced model (5-19 atoms) of the interacting species with a larger model (127-131 atoms) including five amino acids of GST, we show that the protein environment effects must be taken into account to properly model the active site. We find that, in the case of the Tyr --> Phe mutant, the experimental data on pK are reproduced, assuming that a water molecule interacts with the thiol group of GSH. Our results help to elucidate the role that Tyr and water may play as H-bond donors to the thiol group in the enzymatic reaction of GST.

Electronic structure and hyperfine interactions in thioether-substituted tyrosyl radicals

Electron-magnetic resonance spectroscopy and computational studies of the cysteine cross-linked tyrosyl radical in apogalactose oxidase have led to conflicting ideas regarding spectral assignments and protein-environment effects. We report DFT calculations on model radicals that clarify these issues. Calculated Fermi contact interactions do not resolve the ambiguity in spectral assignments; better insight is provided by the anisotropy of the hyperfine interactions and the systematic effects of thioether substitution. DFT results on model systems do not account for salient properties of the apogalactose radical. This inadequacy suggests that the protein environment exerts significant effects on the electronic structure of the radical.

The Effects of Protein Environment on the Low Temperature Electronic Spectroscopy of Cytochrome c and Microperoxidase-11

Low temperature UV-visible spectra of cytochrome c and microperoxidase-11 are studied experimentally and theoretically using quantum chemical and Poisson−Boltzmann electrostatics models. Spectral s...

Interactions of concanavalin A with glycoproteins. A quantitative precipitation study of concanavalin A with the soybean agglutinin

Certain oligomannose-type glycopeptides have been previously shown to be bivalent for binding to concanavalin A and capable of precipitating the lectin by forming homogeneous cross-linked lattices [L. Bhattacharyya, M. I. Khan, and C. F. Brewer, Biochemistry, 27 (1988) 8762–8767]. In the present study, the effect of protein environment on the binding properties of an oligomannose-type oligosaccharide has been examined through quantitative precipitation analysis of the interactions of concanavalin A (Con A) with the soybean ( Glycine max) agglutinin (SBA), which is a tetrameric glycoprotein possessing a single Man 9-oligomannose chain per monomer. The results showed that SBA forms two different types of cross-linked complexes with tetrameric Con A, depending on the relative ratio of the two molecules in solution. At a concentration of one equivalent or less, SBA forms a 1:1 complex with Con A. At concentrations exceeding one equivalent, SBA forms a 2:1 complex with Con A. However, SBA forms only 1:1 cross-linked complexes with dimeric forms of Con A, such as acetyl- and succinyl-Con A. The results demonstrated that the total valency of the carbohydrate of SBA is a function of both the quaternary structure of Con A, as well as the relative ratio of SBA to Con A. In addition, the individual Man 9-oligosaccharide, which as a glycopeptide is bivalent for binding to Con A, expresses univalency when present on the protein matrix of SBA.

Solid-state carbon-13 NMR study of tyrosine protonation in dark-adapted bacteriorhodopsin

Solid-state 13C MAS NMR spectra were obtained for dark-adapted bacteriorhodopsin (bR) labeled with [4'-13C]Tyr. Difference spectra (labeled minus natural abundance) taken at pH values between 2 and 12, and temperatures between 20 and -90 degrees C, exhibit a single signal centered at 156 ppm, indicating that the 11 tyrosines are protonated over a wide pH range. However, at pH 13, a second line appears in the spectrum with an isotropic shift of 165 ppm. Comparisons with solution and solid-state spectra of model compounds suggest that this second line is due to the formation of tyrosinate. Integrated intensities indicate that about half of the tyrosines are deprotonated at pH 13. This result demonstrates that deprotonated tyrosines in a membrane protein are detectable with solid-state NMR and that neither the bR568 nor the bR555 form of bR present in the dark-adapted state contains a tyrosinate at pH values between 2 and 12. Deprotonation of a single tyrosine in bR568 would account for 3.6% of the total tyrosine signal, which would be detectable with the current signal-to-noise ratio. We observe a slight heterogeneity and subtle line-width changes in the tyrosine signal between pH 7 and pH 12, which we interpret to be due to protein environmental effects (such as changes in hydrogen bonding) rather than complete deprotonation of tyrosine residue(s).