Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Quantum chemical (QM:MM) investigation of the mechanism of enzymatic reaction of tryptamine and N,N-dimethyltryptamine with monoamine oxidase A.

The endogenous psychedelic (mind-altering) N,N-dimethyltryptamine (DMT) molecule has an important role in tissue protection, regeneration, and immunity via sigma-1 receptor activation as its natural ligand. The immunologic properties of DMT suggest this biogenic compound should be investigated thoroughly in other aspects as well. In our in silico project, we examined the metabolism of DMT and its primary analogue, the tryptamine (T), by the monoamine oxidase (MAO) flavoenzyme. MAO has two isoforms, MAO-A and MAO-B. MAOs perform the oxidation of various monoamines by their flavin adenine dinucleotide (FAD) cofactor. Two-layer QM:MM calculations at the ONIOM(M06-2X/6-31++G(d,p):UFF=QEq) level were performed including the whole enzyme to explore the potential energy surface (PES) of the reactions. Our findings reinforced that a hybrid mechanism, a mixture of pure H+ and H- transfer pathways, describes precisely the rate-determining step of amine oxidation as suggested by earlier works. Additionally, our results show that the oxidation of tertiary amine DMT requires a lower activation barrier than the primary amine T. This may reflect a general rule, thus we recommend further investigations. Furthermore, we demonstrated that at pH 7.4 the protonated form of these substrates enter the enzyme. As the deprotonation of substrates is crucial, we presumed protonated cofactor, FADH+, may form. Surprisingly, the activation barriers are much lower compared to FAD with both substrates. Therefore, we suggest further investigations in this direction.

Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles.

Phenylalanine ammonia-lyase (PAL), found in many organisms, catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in-line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)-pent-2-ene-4-ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel-Crafts-type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)-pent-2-ene-4-ynoate yielding enantiopure l-PG, contradicts the proposed highly exothermic single-step mechanism. Computations with the QM/MM models of the N-MIO intermediates from L-PG and L-Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N-MIO intermediate.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Mechanism of the tyrosine ammonia lyase reaction-tandem nucleophilic and electrophilic enhancement by a proton transfer.

Quantum mechanics/molecular mechanics calculations in tyrosine ammonia lyase (TAL) ruled out the hypothetical Friedel-Crafts (FC) route for ammonia elimination from L-tyrosine due to the high energy of FC intermediates. The calculated pathway from the zwitterionic L-tyrosine-binding state (0.0 kcal mol(-1)) to the product-binding state ((E)-coumarate+H(2)N-MIO; -24.0 kcal mol(-1); MIO = 3,5-dihydro-5-methylidene-4H-imidazol-4-one) involves an intermediate (IS, -19.9 kcal mol(-1)), which has a covalent bond between the N atom of the substrate and MIO, as well as two transition states (TS1 and TS2). TS1 (14.4 kcal mol(-1)) corresponds to a proton transfer from the substrate to the N1 atom of MIO by Tyr300-OH. Thus, a tandem nucleophilic activation of the substrate and electrophilic activation of MIO happens. TS2 (5.2 kcal mol(-1)) indicates a concerted C-N bond breaking of the N-MIO intermediate and deprotonation of the pro-S ß position by Tyr60. Calculations elucidate the role of enzymic bases (Tyr60 and Tyr300) and other catalytically relevant residues (Asn203, Arg303, and Asn333, Asn435), which are fully conserved in the amino acid sequences and in 3D structures of all known MIO-containing ammonia lyases and 2,3-aminomutases.

Antibody recognition and conformational flexibility of a plaque-specific beta-amyloid epitope modulated by non-native peptide flanking regions.

Here we report on the synthesis, antibody binding, and QSAR studies of a series of linear and cyclic peptides containing a beta-amyloid plaque-specific epitope (Abeta(4-10); FRHDSGY). In these constructs, two or three alpha- l-Ala, alpha- d-Ala, or beta-Ala residues were introduced at both N- and C-termini of the epitope as non-native flanking sequences. Cyclization of the linear Abeta(4-10) epitope peptide resulted in reduced antibody binding. However, the antibody binding could be fully compensated by insertion of alanine flanks into the corresponding cyclic peptides. These results indicate that the modification of a beta-amyloid plaque-specific epitope by combination of cyclization and flanking sequences could generate highly antigenic peptides compared to the native sequence. A novel 3D QSAR method, which explicitly handles conformational flexibility, was developed for the case of such molecular libraries. This method led to the prediction of the binding conformation for the common FRHDSGY sequence.

Effect of substituents on activation parameters in aliphatic S(N)2 reactions. A DFT study.

The activation parameters and optimized structures of the reactants and transition states in the S(N)2 reactions of substituted pyridines and N,N-dimethylanilines with methyl iodide were computed at the DFT level in different solvents. The measured and calculated deltaG/deltaH/deltaS versus sigma plots proved to be linear, and their slopes, the deltadeltaG, deltadeltaH, and deltadeltaS reaction constants, were determined. The least solvent-dependent deltadeltaG reaction constants can be computed with acceptable accuracy. The calculated deltadeltaS data decrease only very slightly with the jointly increasing electron-withdrawing effect of the substituents and tightness of the transition states. The measured deltadeltaS values are influenced mainly by the change of solvation in the reactions, and deltadeltaH is also influenced by the reorganization of the solvent. Consequently, the experimental and calculated deltadeltaS and deltadeltaH reaction constants may deviate considerably from each other. In dipolar aprotic solvents the measured deltadeltaS was less than zero, and in protic solvents it was greater than zero. The ordering of the solvent molecules around the transition state with increasing charge is increased in the former but decreased in the latter media, as compared to the bulk of the solvents. The calculated deltaG(o), deltaH(o), and deltaS(o) parameters of the unsubstituted compounds agree relatively well with the experimental data for reactions of neutral molecules in dipolar aprotic solvents (e.g., XC6H4N(CH3)2 + CH3I). On the other hand, the measured and calculated activation parameters may show considerable deviations for reactions of ions (e.g., XC5H4NCH3+ + I-) and for any reaction in protic solvents.

Vibrations-determined properties of green fluorescent protein.

The physicochemical characteristics of the green fluorescent protein (GFP), including the thermodynamic properties (entropy, enthalpy, Gibbs' free energy, heat capacity), normal mode vibrations, and atomic fluctuations, were investigated. The Gaussian 03 computational chemistry program was employed for normal mode analysis using the AMBER force field. The thermodynamic parameters and atomic fluctuations were then calculated from the vibrational eigenvalues (frequencies) and eigenvectors. The regions of highest rigidity were shown to be the beta-sheet barrel with the central alpha-helix, which bears the chromophore. The most flexible parts of the GFP molecule were the outlying loops that cover the top and bottom of the beta-barrel. This way, the balance between rigidity and flexibility is maintained, which is the optimal relationship for protein stability in terms of Gibbs' free energy. This dual-schemed structure satisfies the requirements for GFP function. In this sense, the structure of GFP resembles a nanoscale drum: a stiff cylinder with flexible vibrating end(s).

Peptide models. XXXIII. Extrapolation of low-level Hartree-Fock data of peptide conformation to large basis set SCF, MP2, DFT, and CCSD(T) results. The Ramachandran surface of alanine dipeptide computed at various levels of theory.

At the dawn of the new millenium, new concepts are required for a more profound understanding of protein structures. Together with NMR and X-ray-based 3D-structure determinations in silico methods are now widely accepted. Homology-based modeling studies, molecular dynamics methods, and quantum mechanical approaches are more commonly used. Despite the steady and exponential increase in computational power, high level ab initio methods will not be in common use for studying the structure and dynamics of large peptides and proteins in the near future. We are presenting here a novel approach, in which low- and medium-level ab initio energy results are scaled, thus extrapolating to a higher level of information. This scaling is of special significance, because we observed previously on molecular properties such as energy, chemical shielding data, etc., determined at a higher theoretical level, do correlate better with experimental data, than those originating from lower theoretical treatments. The Ramachandran surface of an alanine dipeptide now determined at six different levels of theory [RHF and B3LYP 3-21G, 6-31+G(d) and 6-311++G(d,p)] serves as a suitable test. Minima, first-order critical points and partially optimized structures, determined at different levels of theory (SCF, DFT), were completed with high level energy calculations such as MP2, MP4D, and CCSD(T). For the first time three different CCSD(T) sets of energies were determined for all stable B3LYP/6-311++G(d,p) minima of an alanine dipeptide. From the simplest ab initio data (e.g., RHF/3-21G) to more complex results [CCSD(T)/6-311+G(d,p)//B3LYP/6-311++G(d,p)] all data sets were compared, analyzed in a comprehensive manner, and evaluated by means of statistics.

Geometry optimization with QM/MM, ONIOM, and other combined methods. I. Microiterations and constraints.

Hybrid energy methods such as QM/MM and ONIOM, that combine different levels of theory into one calculation, have been very successful in describing large systems. Geometry optimization methods can take advantage of the partitioning of these calculations into a region treated at a quantum mechanical (QM) level of theory and the larger, remaining region treated by an inexpensive method such as molecular mechanics (MM). A series of microiterations can be employed to fully optimize the MM region for each optimization step in the QM region. Cartesian coordinates are used for the MM region and are chosen so that the internal coordinates of the QM region remain constant during the microiterations. The coordinates of the MM region are augmented to permit rigid body translation and rotation of the QM region. This is essential if any atoms in the MM region are constrained, but it also improves the efficiency of unconstrained optimizations. Because of the microiterations, special care is needed for the optimization step in the QM region so that the system remains in the same local valley during the course of the optimization. The optimization methodology with microiterations, constraints, and step-size control are illustrated by calculations on bacteriorhodopsin and other systems.

Effects of the protein environment on the structure and energetics of active sites of metalloenzymes. ONIOM study of methane monooxygenase and ribonucleotide reductase.

As the first application of our recently developed ONIOM2(QM:MM) and ONIOM3(QM:QM:MM) codes to the metalloenzymes with a large number of protein residues, two members of the non-heme protein family, methane monooxygenause and ribonucleotide reductase, have been chosen. The "active-site + four alpha-helical fragments" model was adopted which includes about 1000 atoms from 62 residues around the Fe-centered spheres. Comparison of the active-site geometries of MMOH and R2 units optimized with this model with those obtained with the "active site only" (with only 39-46 atoms) model and the X-ray results clearly demonstrates the crucial role of the active site-protein interaction in the enzymatic activities.