Structure and solvation of melittin in 1,1,1,3,3,3-hexafluoro-2-propanol/water

Fluorinated alcohols can induce peptides and proteins to take up helical conformations. Nuclear Overhauser effect (NOE) spectroscopy experiments and analysis of C(alpha)H proton chemical shifts show that the conformation of melittin in 35% hexafluoro-2-propanol/water is alpha-helical from residues Ile-2 to Val-8 and from Leu-13 to Gln-25. As has been found in other solvent systems, the two helical regions are not colinear; the interhelix angle (73 +/- 15 degrees ) in 35% 1,1,1,3,3,3-hexafluoro-2-propanol/water is smaller than the angle found in other fluoroalcohol-water mixtures or in the crystal. Intermolecular (1)H(19)F and (1)H(1)H nuclear Overhauser effects were used to explore interaction of solvent components with melittin dissolved in this solvent mixture. The NOEs observed indicate that fluoroalcohol and water molecules are both tightly bound to the peptide in the vicinity of the interhelix bend. For the remainder of the molecule, solute-solvent NOEs are consistent with preferential solvation of the peptide by the fluoroalcohol component of the solvent mixture.     

Structure and solvation of melittin in hexafluoroacetone/water

Intermolecular (1)H[(19)F] and (1)H[(1)H] nuclear Overhauser effects have been used to explore interaction of solvent components with melittin dissolved in 50% hexafluoroacetone trihydrate (HFA)/water. Standard nuclear Overhauser effect experiments and an analysis of C(alpha)H proton chemical shifts confirm that the conformation of the peptide in this solvent is alpha-helical from residues Ala4 to Thr11 and from Leu13 to Arg24. The two helical regions are not collinear; the interhelix angle (144 +/- 20 degrees ) found in this work is near that observed in the solid state and previous NMR studies. Intermolecular NOEs arising from interactions between spins of the solvent and the solute indicate that both fluoroalcohol and water molecules are strongly enough bound to the peptide that solvent-solute complexes persist for > or =2 ns. Preferential interactions of HFA with many hydrophobic side chains of the peptide are apparent while water molecules appear to be localized near hydrophilic side chains. These results indicate that interactions of both HFA and water are qualitatively different from those present when the peptide is dissolved in 35% hexafluoro-2-propanol/water, a chemically similar helix-supporting solvent system.     

Interactions of hexafluoro-2-propanol with the Trp-cage peptide

Fluoro alcohols present in aqueous solutions can alter the dominant conformations of peptides and proteins. The origins of these effects likely are related to the details of solute-fluoro alcohol interactions. Preferential interaction of the fluoro alcohol component of a fluoro alcohol-water mixture with peptide solutes has been demonstrated by several experimental approaches. In the present work, we have used 1H{19F} intermolecular NOE experiments to examine interactions of hexafluoro-2-propanol in a 30% fluoro alcohol-50 mM phosphate buffer solvent mixture with the "Trp-cage" peptide (NLY IQW LKD GGP SSG RPP PS). The results show that the peptide is selectively solvated by hexafluoro-2-propanol to the extent that the fluoro alcohol concentration near the peptide may be 3 to 4 times higher than the nominal concentration of fluoro alcohol in the bulk sample. The observed NOEs indicate that peptide-fluoro alcohol interactions persist for times of the order of 1 ns at 5 degrees C. As the sample temperature is increased, the lifetimes of fluoro alcohol interactions with several exposed side chains decrease to the extent that the peptide hydrogen-solvent fluorine interactions appear to become diffusive in nature, with interaction lifetimes of approximately 0.03 ns. It is known that protein molecules can provide specific sites for binding small organic solvent molecules. Our work suggests that small peptides also have this ability and that the dynamics for such interactions can be site-specific.     

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.     

Application of 13C(omega 1)-half-filtered [1H,1H]-NOESY for studies of a complex formed between DNA and a 13C-labeled minor-groove-binding drug

The complex formed between the anticancer drug 4-[p-[p-(4-quinolylamino)benzamido]anilino]pyridine (SN 6999) and the decadeoxyribonucleoside nonaphosphate d-(GCATTAATGC)2 was investigated using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) with a 13C(omega 1)-half-filter. The two quaternary methyl groups in SN 6999 had been labeled with 13C for these experiments. The simplified subspectra of [1H,1H]-NOESY obtained with this procedure greatly facilitate the identification and assignment of intermolecular NOEs. Quite generally, the combined use of isotope labeling and heteronuclear filters in [1H,1H]-NOESY provides an improved experimental basis for structural studies of drug/DNA complexes.     

Solvent reorganization contribution to the transfer thermodynamics of small nonpolar molecules.

The experimental thermodynamic data for the dissolution of five simple hydrocarbon molecules in water were combined with the solute-solvent interaction energy from a computer simulation study to yield data on the enthalpy change of solvent reorganization. Similar data were generated for dissolving these same solute molecules in their respective neat solvents using the equilibrium vapor pressure and the heat of vaporization data for the pure liquid. The enthalpy and the free energy changes upon cavity formation were also estimated using the temperature dependence of the solute-solvent interaction energy. Both the enthalpy and T delta S for cavity formation rapidly increase with temperature in both solvent types, and the free energy of cavity formation can be reproduced accurately by the scaled particle theory over the entire temperature range in all cases. These results indicate that the characteristic structure formation around an inert solute molecule in water produces compensating changes in enthalpy and entropy, and that the hydrophobicity arises mainly from the difference in the excluded volume effect.     

Conformations of dehydrophenylalanine containing peptides: nmr studies of an acyclic hexapeptide with two delta Z-Phe residues

The conformation of an acyclic dehydrophenylalanine (delta Z-Phe) containing hexapeptide, Boc-Phe-delta Z-Phe-Val-Phe-delta Z-Phe-Val-OMe, has been investigated in CDCl3 and (CD3)2SO by 270-MHz 1H-nmr. Studies of NH group solvent accessibility and observation of interresidue nuclear Overhauser effects (NOEs) suggest a significant solvent-dependent conformational variability. In CDCl3, a population of folded helical conformations is supported by the inaccessibility to solvent of the NH groups of residues 3-6 and the detection of several NiH----Ni + 1H NOEs. Evidence is also obtained for conformational heterogeneity from the detection of some Ci alpha H----Ni + 1H NOEs characteristic of extended strands. In (CD3)2SO, the peptide largely favors an extended conformation, characterized by five solvent-exposed NH groups and successive Ci alpha H----Ni + 1H NOEs for the L-residues and Ci beta H----Ni + 1H NOEs for the delta Z-Phe residues. The results suggest that delta Z-Phe residues do not provide compelling conformational constraints.     

A combined spectroscopic and TDDFT investigation of the solute‐solvent interactions of two coumarin derivatives

The UV/Vis absorption and fluorescence characteristics of 3‐cyano‐7‐hydroxycoumarin [ CHC ] and 7‐amino‐4‐methyl‐3‐coumarinylacetic acid [ AMCA‐H ] were studied at room temperature in several neat solvents and binary solvent mixtures of 1,4‐dioxane/acetonitrile. The effects of solvent on the spectral properties are analyzed using single and multi‐parameter solvent polarity scales. Both general solute/solvent interactions and hydrogen bond interactions are operative in these systems. The solvation of CHC and AMCA‐H dyes in 1,4‐dioxane/acetonitrile solvent mixtures has been studied. The solutes CHC and AMCA‐H are preferentially solvated by acetonitrile and a synergistic effect is observed for both molecules in dioxane/acetonitrile solvent mixtures. In addition, using the solvatochromic method the ground‐ and the excited‐state dipole moments of both the dyes were calculated. The ground‐ and excited‐state dipole moments, absorption and emission maxima and HOMO–LUMO gap were also estimated theoretically using B3LYP/6–311+ G (d,p) level of theory in the gaseous phase, dioxane and acetonitrile solvents. Furthermore, changes in dipole moment values were also calculated using the variation of Stokes shift with the molecular–microscopic empirical solvent polarity parameter ( ). The observed excited‐state dipole moments are larger than their ground‐state counterparts, indicating a substantial redistribution of the electron densities in a more dipolar excited state for both coumarins investigated.     

Backbone dynamics of reduced plastocyanin from the cyanobacterium Anabaena variabilis: regions involved in electron transfer have enhanced mobility

The dynamics of the backbone of the electron-transfer protein plastocyanin from the cyanobacterium Anabaena variabilis were determined from the (15)N and (13)C(alpha) R(1) and R(2) relaxation rates and steady-state [(1)H]-(15)N and [(1)H]-(13)C nuclear Overhauser effects (NOEs) using the model-free approach. The (13)C relaxation studies were performed using (13)C in natural abundance. Overall, it is found that the protein backbone is rigid. However, the regions that are important for the function of the protein show moderate mobility primarily on the microsecond to millisecond time scale. These regions are the "northern" hydrophobic site close to the metal site, the metal site itself, and the "eastern" face of the molecule. In particular, the mobility of the latter region is interesting in light of recent findings indicating that residues also on the eastern face of plastocyanins from prokaryotes are important for the function of the protein. The study also demonstrates that relaxation rates and NOEs of the (13)C(alpha) nuclei of proteins are valuable supplements to the conventional (15)N relaxation measurements in studies of protein backbone dynamics.     

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motion

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.     

Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited 1H NMR and site-directed mutagenesis.

The binary complex of diacetylchloramphenicol and chloramphenicol acetyltransferase (CAT) has been studied by a combination of isotope-edited 1H NMR spectroscopy and site-directed mutagenesis. One-dimensional HMQC spectra of the complex between 1,3-[2-13C]diacetylchloramphenicol and the type III natural variant of CAT revealed the two methyl 1H signals arising from each 13C-labeled carbon atom in the acetyl groups of the bound ligand. Slow hydrolysis of the 3-acetyl group by the enzyme precluded further analysis of this binary complex. It was possible to slow down the rate of hydrolysis by use of the catalytically defective S148A mutant of CATIII (Lewendon et al., 1990); in the complex of diacetylchloramphenicol with S148A CATIII, the chemical shifts of the acetyl groups of the bound ligand were the same as in the wild-type complex. The acetyl signals were individually assigned by repeating the experiment using 1-[2-13C],3-[2-12C]diacetylchloramphenicol, where only one signal from the bound ligand was observed. A two-dimensional 1H, 1H NOESY experiment, with 13C(omega 2) half-filter, on the 1,3-[2-13C]diacetylchloramphenicol/S148A CATIII complex showed a number of intermolecular NOEs from each methyl group in the ligand to residues in the chloramphenicol binding site. The 3-acetyl group showed strong NOEs to two aromatic signals which were selected for assignment. The possibility that the NOEs originated from the aromatic protons of diacetylchloramphenicol itself was eliminated by assignment of the signals from enzyme-bound diacetylchloramphenicol and chloramphenicol using perdeuterated CATIII. Examination of the X-ray crystal structure of the chloramphenicol/CATIII binary complex indicated four plausible candidate aromatic residues: Y25, F33, F103, and F158.(ABSTRACT TRUNCATED AT 250 WORDS)     

The non-polar solvent potential of mean force for the dimerization of alanine dipeptide: the role of solute-solvent van der Waals interactions

The non-polar component of the potential of mean force of dimerization of alanine dipeptide has been calculated in explicit solvent by free energy perturbation. We observe that the calculated PMF is inconsistent with a non-polar hydration free energy model based solely on the solute surface area. The non-linear behavior of the solute-solvent van der Waals energy is primarily responsible for the non-linear dependence of the potential of mean force with respect to the surface area. The calculated potential of mean force is reproduced by an implicit solvent model based on a solvent continuum model for the solute-solvent van der Waals interaction energy and the surface area for the work of forming the solute cavity.     

Solution conformation of a tetradecapeptide stabilized by two di‐n‐propyl glycine residues

The solution conformation of a designed tetradecapeptide Boc‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐OMe (Dpg‐14) containing two di‐n‐propyl glycine (Dpg) residues has been investigated by ¹H NMR and circular dichroism in organic solvents. The peptide aggregates formed at a concentration of 3 mM in the apolar solvent CDCl₃ were broken by the addition of 12% v/v of the more polar solvent DMSO‐d₆. Successive N_iH N_i+1H NOEs observed over the entire length of the sequence in this solvent mixture together with the observation of several characteristic medium‐range NOEs support a major population of continuous helical conformations for Dpg‐14. Majority of the observed coupling constants ( ) also support ? values in the helical conformation. Circular dichroism spectra recorded in methanol and propan‐2‐ol give further support in favor of helical conformation for Dpg‐14 and the stability of the helix at higher temperature. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

Radiosynthesis of the tumor hypoxia marker [18F]TFMISO via O-[18F]trifluoroethylation reveals a striking difference between trifluoroethyl tosylate and iodide in regiochemical reactivity toward oxygen nucleophiles

The MRI hypoxia marker trifluoromisonidazole (TFMISO) [1-(2-nitro-1H-imidazol-1-yl)-3-(2,2,2-trifluoroethoxy)propan-2-ol] was successfully labeled with (18)F to expand its role into a bimodal PET/MRI probe. (18)F-Labeling was achieved via a three-step procedure in which 2,2,2-[(18)F]trifluoroethyl p-toluenesulfonate prepared by (18)F-(19)F exchange served as the [(18)F]trifluoroethylating agent. The O-[(18)F]trifluoroethylation reaction proceeded efficiently to give the intermediate 1,2-epoxy-3-(2,2,2-[(18)F]trifluoroethoxy)propane, with approximately 60% of (18)F incorporated from the tosylate precursor, which was condensed with 2-nitroimidazole to yield [(18)F]TFMISO. Approximately 40% of the [(18)F]trifluoroethyl tosylate precursor was converted into the final product. In stark contrast, 2,2,2-[(18)F]trifluoroethyl iodide failed to produce [(18)F]TFMISO, giving instead 1,1-[(18)F]difluoro-2-iodoethoxy and 1-[(18)F]fluoro-2-iodovinyloxy analogs of [(18)F]TFMISO. Thus, this investigation has identified 2,2,2-[(18)F]trifluoroethyl tosylate as an excellent [(18)F]trifluoroethylating agent, which can convert efficiently an alcohol into the corresponding [(18)F]trifluoroethyl ether.     

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.     

Size effects in nonpolar solvation: lessons from two simple models

Size dependence of the solute chemical potential mu(u) is examined using the Ornstein-Zernike equation for two models of the nonpolar solute-solvent interactions. Simple Lennard-Jones interactions are assumed in the first model while the Lennard-Jones potential is distributed over the solute volume in the second model similar to the Hamaker theory for the colloid dispersion forces. In both models, while mu(u) rises asymptotically as the third power of the solute size in agreement with asymptotic solution of the scaled particle theory, it increases faster at smaller sizes. Deviations from the cubic law are more pronounced at higher solvent densities and stronger molecular interactions. Within a relatively narrow size range typical for small organic molecules, mu(u) can be approximated with a polynomial of the third or even the second power. However, the latter approximation is less accurate and cannot be employed for extrapolation to the larger size region.     

Some NMR Experiments and a Structure Determination Employing a {15N,2H} Enriched Protein

We present the results of studies of an aqueous sample of a highly {15N,2H} enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7Å apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.