Conformational analysis of dehydrodidemnin B (aplidine) by NMR spectroscopy and molecular mechanics/dynamics calculations.

Dehydrodidemnin B (DDB or aplidine), a potent antitumoral natural product currently in phase II clinical trials, exists as an approximately 1:1 mixture of two slowly interconverting conformations. These are sufficiently long-lived so as to allow their resolution by HPLC. NMR spectroscopy shows that this phenomenon is a consequence of restricted rotation about the Pyr-Pro(8) terminal amide bond of the molecule's side chain. The same technique also indicates that the overall three-dimensional structures of both the cis and trans isomers of DDB are similar despite the conformational change. Molecular dynamics simulations with different implicit and explicit solvent models show that the ensembles of three-dimensional structures produced are indeed similar for both the cis and trans isomers. These studies also show that hydrogen bonding patterns in both isomers are alike and that each one is stabilized by a hydrogen bond between the pyruvyl unit at the terminus of the molecule's side chain and the Thr(6) residue situated at the junction betwen the macrocycle and the molecule's side chain. Nevertheless, each conformational isomer forms this hydrogen bond using a different pyruvyl carbonyl group: CO(2) in the case of the cis isomer and CO(1) in the case of the trans isomer.     

Conformational analysis of thia crown ether derivatives by NMR spectroscopy and molecular mechanics calculations

Summary Addition of sulfur dichloride to tetrachlorocatechol-bisallylether (1) yields the 9- and 10-ring thia crown ether derivatives2 and3, respectively, together with the dithia-18-crown-6-ether4. The 10-membered ring compound3 represents the first thia macrocycle containing bothMarkovnikov andanti-Markovnikov constitution of the -chloro-thio structural segments in the same molecule. By¹H and¹³C NMR spectroscopy, equal amounts of two preferred conformers of the only isolated diastereomer of3 were observed at temperatures below –50°C. The signals were assigned to these conformers using COSY, HETCOR, and phase sensitive NOESY spectra at low temperatures. The preferred conformations and the relative configuration were determined using the different effects of _gauche -and _anti -positions in¹³C NMR chemical shifts and analyzing vicinal³ J _H,H coupling constants. These results were confirmed by molecular mechanics calculations.Dedicated to Prof. Dr.Rolf Borsdorf on the occasion of his 65^th birthday     

Analysis of lipoproteins using 2D diffusion-edited NMR spectroscopy and multi-way chemometrics

This study represents the first application of multi-way calibration by N-PLS and multi-way curve resolution by PARAFAC to 2D diffusion-edited ¹H NMR spectra. The aim of the analysis was to evaluate the potential for quantification of lipoprotein main- and subfractions in human plasma samples. Multi-way N-PLS calibrations relating the methyl and methylene peaks of lipoprotein lipids to concentrations of the four main lipoprotein fractions as well as 11 subfractions were developed with high correlations (R = 0.75-0.98). Furthermore, a PARAFAC model with four chemically meaningful components was calculated from the 2D diffusion-edited spectra of the methylene peak of lipids. Although the four extracted PARAFAC components represent molecules of sizes that correspond to the four main fractions of lipoproteins, the corresponding concentrations of the four PARAFAC components proved not to be correlated to the reference concentrations of these four fractions in the plasma samples as determined by ultracentrifugation. These results indicate that NMR provides complementary information on the classification of lipoprotein fractions compared to ultracentrifugation.     

Analysis of conformational polymorphism in pharmaceutical solids using solid-state NMR and electronic structure calculations

A detailed analysis of molecular structure in three polymorphic forms of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile is made using a combination of multidimensional solid-state NMR (SSNMR) experiments and molecular modeling via electronic structure calculations. These compounds, collectively referred to as ROY because of their red, orange, and yellow colors, share a similar molecular structure with the exception of the dihedral angle between the phenyl and thiophene rings. The ROY materials make it possible to study the influence of nearly a single degree of freedom on the associated NMR spectra. Using the 2D PASS (Antzutkin et al. J. Magn. Reson. A 1995, 115, 7) experiment, spectral editing techniques, and DFT-based calculations of the local fields, an analysis is made of the sensitivity of all carbon and nitrogen sites to changing molecular conformation. Chemical shift and dipolar coupling information obtained from these experiments vary noticeably between forms and are subsequently used to quantitatively determine aspects of molecular structure in these materials, including the coplanar angle between the phenyl and thiophene rings. The influence of motion on the methyl and nitro chemical shifts is also investigated. The accuracy of the information obtained from local field analysis and the model structure calculation demonstrates the capabilities of SSNMR as a quantitative structural method.     

Investigation of the dynamics of the conformational transitions of dimeric isoquinoline alkaloids by nmr spectroscopy and molecular mechanics

The conformational dynamics of the dimeric isoquinoline alkaloid turconidine has been investigated by the methods of NMR spectroscopy and molecular mechanics. The main conformational states and the trajectories of their mutual transitions have been established. A high lability of the molecule and the predominant state of the dimer in three of the eight possible conformations with respect to the ether bond have been shown.Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 285–289, March-April, 1995. Original article submitted November 28, 1994.     

High-performance selective excitation pulses for solid- and liquid-state NMR spectroscopy.

Computer-optimized selective pulses are routinely used in solution-state NMR spectroscopy. At the same time, their utility and importance for solid-state applications has yet to be fully realized. We suggest a new computational approach that makes the design of soft selective pulses with desired properties relatively straightforward. By applying this technique to the generic selective excitation problem, we have arrived at a family of high performance selective excitation pulses, dubbed E-Family, that allows more flexibility and better performance than analogous pulses previously reported in the literature. The new pulses have been successfully tested in both solid- and solution-state NMR experiments. A theoretical treatment of the effects of chemical shift anisotropy (CSA) on the selective excitation in magic-angle spinning (MAS) experiments in solids is presented. The set of heuristics that comprise our new strategy were incorporated into a general NMR simulation program SPINEVOLUTION.     

Using molecular dynamics and quantum mechanics calculations to model fluorescence observables

We provide a critical examination of two different methods for generating a donor-acceptor electronic coupling trajectory from a molecular dynamics (MD) trajectory and three methods for sampling that coupling trajectory, allowing the modeling of experimental observables directly from the MD simulation. In the first coupling method we perform a single quantum-mechanical (QM) calculation to characterize the excited state behavior, specifically the transition dipole moment, of the fluorescent probe, which is then mapped onto the configuration space sampled by MD. We then utilize these transition dipoles within the ideal dipole approximation (IDA) to determine the electronic coupling between the probes that mediates the transfer of energy. In the second method we perform a QM calculation on each snapshot and use the complete transition densities to calculate the electronic coupling without need for the IDA. The resulting coupling trajectories are then sampled using three methods ranging from an independent sampling of each trajectory point (the independent snapshot method) to a Markov chain treatment that accounts for the dynamics of the coupling in determining effective rates. The results show that the IDA significantly overestimates the energy transfer rate (by a factor of 2.6) during the portions of the trajectory in which the probes are close to each other. Comparison of the sampling methods shows that the Markov chain approach yields more realistic observables at both high and low FRET efficiencies. Differences between the three sampling methods are discussed in terms of the different mechanisms for averaging over structural dynamics in the system. Convergence of the Markov chain method is carefully examined. Together, the methods for estimating coupling and for sampling the coupling provide a mechanism for directly connecting the structural dynamics modeled by MD with fluorescence observables determined through FRET experiments.     

Fast 2D NMR ligand screening using Hadamard spectroscopy

Fast 2D NMR-based screening can be achieved using Hadamard encoded spectroscopy to focus on the signals of interest (e.g., enzyme active or ligand recognition sites). By recording a set of Hadamard spectra (a "Hadamard constellation") with relative offsets comparable to the excitation bandwidth, quantitative ligand-induced shifts can be obtained from peak intensities.     

Analytical calculations using a computer in molecular spectroscopy

The paper deals with the discussion of problems of calculating the vibration-rotation energy and wave functions of polyatomic molecules using systems for analytical calculations. The system developed can be used for obtaining the analytical expressions for the spectroscopic parameters which are useful for solving both the direct and inverse spectroscopic problems.     

A study on the anisole-water complex by molecular beam-electronic spectroscopy and molecular mechanics calculations

An experimental and theoretical study is made on the anisole-water complex. It is the first van der Waals complex studied by high resolution electronic spectroscopy in which the water is seen acting as an acid. Vibronically and rotationally resolved electronic spectroscopy experiments and molecular mechanics calculations are used to elucidate the structure of the complex in the ground and first electronic excited state. Some internal dynamics in the system is revealed by high resolution spectroscopy.     

Protonation and conformational dynamics of GFP mutants by two-photon excitation fluorescence correlation spectroscopy

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.     

Study of molecular dynamics and the solid state phase transition mechanism for unsymmetrical thiopyrophosphate using X-ray diffraction, DFT calculations and NMR spectroscopy

Differential scanning calorimetry (DSC) and low-temperature X-ray diffraction studies showed that 2-thio-(5,5-dimethyl-1,3,2-dioxaphosphorinanyl)2'-oxo-dineopentyl-thiophosphate (compound 1) undergoes reversible phase transition at 203 K related to the change of symmetry of the crystallographic unit. Solid state NMR spectroscopy was used to establish the dynamic processes of aliphatic groups and the phosphorus skeleton. 13C and 31P variable temperature NMR studies as well as T1 and T1rho measurements of relaxation times revealed the different mode of molecular motion for each neopentyl residue directly bonded to phosphorus. It is concluded that molecular dynamics of aliphatic groups causes different van der Waals interactions in the crystal lattice and is the driving force of phase transition for compound 1. Finally, we showed that very sharp phase transition temperature makes compound 1 an excellent candidate as a low-temperature NMR thermometer in the solid phase.     

Exploring multiple timescale motions in protein GB3 using accelerated molecular dynamics and NMR spectroscopy

Biological function relies on the complex spectrum of conformational dynamics occurring in biomolecules. We have combined Accelerated Molecular Dynamics (AMD) with experimental results derived from NMR to probe multiple time-scale motions in the third IgG-binding domain of Protein G (GB3). AMD is shown to accurately reproduce the amplitude and distribution of slow motional modes characterized using residual dipolar couplings, reporting on dynamics up to the millisecond timescale. In agreement with experiment, larger amplitude slower motions are localized in the beta-strand/loop motif spanning residues 14-24 and in loop 42-44. Principal component analysis shows these fluctuations participating in the primary mode, substantiating the existence of a correlated motion traversing the beta-sheet that culminates in maximum excursions at the active site of the molecule. Fast dynamics were simulated using extensive standard MD simulations and compared to order parameters extracted from 15N relaxation. Notably 60 2-ns fully solvated MD simulations exploring the different conformational substates sampled from AMD resulted in better reproduction of order parameters compared to the same number of simulations starting from the relaxed crystal structure. This illustrates the inherent dependence of protein dynamics on local conformational topology. The results provide rare insight into the complex hierarchy of dynamics present in GB3 and allow us to develop a model of the conformational landscape native to the protein, appearing as a steep sided potential well whose flat bottom comprises multiple similar but discrete conformational substates.     

Structure and ultrafast dynamics of liquid water: a quantum mechanics/molecular mechanics molecular dynamics simulations study

A quantum mechanics/molecular mechanics molecular dynamics simulation was performed for liquid water to investigate structural and dynamical properties of this peculiar liquid. The most important region containing a central reference molecule and all nearest surrounding molecules (first coordination shell) was treated by Hartree-Fock (HF), post-Hartree-Fock [second-order Moller-Plesset perturbation theory (MP2)], and hybrid density functional B3LYP [Becke's three parameter functional (B3) with the correlation functional of Lee, Yang, and Parr (LYP)] methods. In addition, another HF-level simulation (2HF) included the full second coordination shell. Site to site interactions between oxygen-oxygen, oxygen-hydrogen, and hydrogen-hydrogen atoms of all ab initio methods were compared to experimental data. The absence of a second peak and the appearance of a shoulder instead in the gO-O graph obtained from the 2HF simulation is notable, as this feature has been observed so far only for pressurized or heated water. Dynamical data show that the 2HF procedure compensates some of the deficiency of the HF one-shell simulation, reducing the difference between correlated (MP2) and HF results. B3LYP apparently leads to too rigid structures and thus to an artificial slow down of the dynamics.     

Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy

Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450(cam) from Pseudomonas putida on fast time scales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450(cam) and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450(cam), three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450(cam) toward different substrates.     

Fluctuation theory of molecular association and conformational equilibria

General expressions relating the effects of pressure, temperature, and composition on solute association and conformational equilibria using the fluctuation theory of solutions are provided. The expressions are exact and can be used to interpret experimental or computer simulation data for any multicomponent mixture involving molecules of any size and character at any composition. The relationships involve particle-particle, particle-energy, and energy-energy correlations within local regions in the vicinity of each species involved in the equilibrium. In particular, it is demonstrated that the results can be used to study peptide and protein association or aggregation, protein denaturation, and protein-ligand binding. Exactly how the relevant fluctuating properties may be obtained from experimental or computer simulation data are also outlined. It is shown that the enthalpy, heat capacity, and compressibility differences associated with the equilibrium process can, in principle, be obtained from a single simulation. Fluctuation based expressions for partial molar heat capacities, thermal expansions, and isothermal compressibilities are also provided.     

Structural forms of green fluorescent protein by quantum mechanics/molecular mechanics calculations

The molecular modeling of structural forms of the green fluorescent protein (GFP) with the Ser65Thr single-site mutation was performed by the quantum mechanics/molecular mechanics (QM/MM) method. Two model systems were constructed based on the crystallographic structure from the Protein Data Bank (PDB entry code 1EMA.) The model systems differ in the initial protonation state of the side chain of the amino acid residue Glu222 near the chromophore. The atomic coordinates of the protein macromolecule corresponding to the equilibrium geometric configurations were determined by total energy minimization using the QM/MM method within the density functional theory approximation PBE0/cc-pVDZ for the quantum subsystem that consists of the chromophore, a water molecule, and the side chains of Arg96, Glu222, and Ser205, and with the parameters of the AMBER force field for the molecular mechanics subsystem. In the analysis of the results, particular attention was given to the hydrogen bond redistribution in the chromophore-containing region of the protein caused by a change in the protonation state of the chromophore. The results obtained from the model containing the initially protonated side chain of Glu222 suggest a new interpretation of the photophysical processes in the green fluorescent protein.     

Conformational flexibility of the pentasaccharide LNF-2 deduced from NMR spectroscopy and molecular dynamics simulations

Human milk oligosaccharides (HMOs) are important as prebiotics since they stimulate the growth of beneficial bacteria in the intestine and act as receptor analogues that can inhibit the binding of pathogens. The conformation and dynamics of the HMO Lacto-N-fucopentaose 2 (LNF-2), α-L-Fucp-(1 → 4)[β-D-Galp-(1 → 3)]-β-D-GlcpNAc-(1 → 3)-β-D-Galp-(1 → 4)-D-Glcp, having a Lewis A epitope, has been investigated employing NMR spectroscopy and molecular dynamics (MD) computer simulations. 1D (1)H,(1)H-NOESY experiments were used to obtain proton-proton cross-relaxation rates from which effective distances were deduced and 2D J-HMBC and 1D long-range experiments were utilized to measure trans-glycosidic (3)J(CH) coupling constants. The MD simulations using the PARM22/SU01 force field for carbohydrates were carried out for 600 ns with explicit water as solvent which resulted in excellent sampling for flexible glycosidic torsion angles. In addition, in vacuo MD simulations were performed using an MM3-2000 force field, but the agreement was less satisfactory based on an analysis of heteronuclear trans-glycosidic coupling constants. LNF-2 has a conformationally well-defined region consisting of the terminal branched part of the pentasaccharide, i.e., the Lewis A epitope, and a flexible β-D-GlcpNAc-(1 → 3)-β-D-Galp-linkage towards the lactose unit, which is situated at the reducing end. For this β-(1 → 3)-linkage a negative ψ torsion angle is favored, when experimental NMR data is combined with the MD simulation in the analysis. In addition, flexibility on a similar time scale, i.e., on the order of the global overall molecular reorientation, may also be present for the ? torsion angle of the β-D-Galp-(1 → 4)-D-Glcp-linkage as suggested by the simulation. It was further observed from a temperature variation study that some (1)H NMR chemical shifts of LNF-2 were highly sensitive and this study indicates that Δδ/ΔT may be an additional tool for revealing conformational dynamics of oligosaccharides.     

Backbone dynamics of proteins studied by two-dimensional heteronuclear NMR spectroscopy and molecular dynamics simulations

To investigate the backbone dynamics of proteins ¹⁵N longitudinal and transverse relaxation experiments combined with {¹H, ¹⁵N{ NOE measurements together with molecular dynamics simulations were carried out using ribonuclease T₁ and the complex of ribonuclease T₁ with 2′GMP as a model protein. The intensity decay of individual amide cross peaks in a series of (¹H, ¹⁵N)HSQC spectra with appropriate relaxation periods was fitted to a single exponential by using a simplex algorithm in order to obtain ¹⁵N T₁ and T₂ relaxation times. The relaxation times were analyzed in terms of the “model-free” approach introduced by Lipari and Szabo. In addition, a nanosecond molecular dynamics (MD ) simulation of ribonuclease T₁ and its 2′GMP complex in water was carried out. The angular reorientations of the backbone amide groups were classified with several coordinate frames following a transformation of NH vector trajectories. In this study, NH librations and backbone dihedral angle fluctuations were distinguished. The NH bond librations were found to be similar for all amides as characterized by correlation times of librational motions in a subpicosecond scale. The angular amplitudes of these motions were found to be about 10°–12° for out-of-plane displacements and 3°–5° for the in-plane displacement. The contributions from the much slower backbone dihedral angle fluctuations strongly depend on the secondary structure. The dependence of the amplitude of local motion on the residue location in the backbone is in good agreement with the results of NMR relaxation measurements and the X-ray data. The protein dynamics is characterized by a highly restricted local motion of those parts of the backbone with defined secondary structure as well as by a high flexibility in loop regions. Comparison of the MD and NMR data of the free liganded enzyme ribonuclease T₁ clearly indicates a restriction of the mobility within certain regions of the backbone upon inhibitor binding. © 1996 John Wiley & Sons, Inc.