Proline ring conformations corresponding to a bistable jump model from 13C spin-lattice relaxation times

A formalism for extracting the conformations of a proline ring based on the bistable jump model of R. E. London [(1978) J. Am. Chem. Soc. 100 , 2678–2685] from ¹³C spin-lattice relaxation times (T₁) is given. The method is such that the relaxation data are only partially used to generate the conformations; these conformations are constrained to satisfy the rest of the relaxation data and to yield acceptable ring geometry. An alternate equation for T₁ of ¹³C nuclei to that of London is given. The formalism is illustrated through an example.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Conformational and dynamic features of cocaine in DMSO-d6 solution

13C spin-lattice relaxation rates, 13C {1H} NOESs, 1H spin-spin relaxation rates and 1H two-dimensional magnetization transfer spectroscopy were used for delineating conformational features of cocaine in DMSO-d6 solution. Two main conformations differing in the orientation of the plane made by the benzoxy substituent with respect to the piperidine ring principal axis were observed. Relatively slow interconversions of the piperidine ring were delineated together with the main motional features of the whole molecule.     

Molecular mobility of polypeptides containing proline as determined by 13C magnetic resonance

The molecular conformations and dynamics of poly(L -prolyl), poly(hydroxyl-L -prolyl), poly(L -prolyl-glycyl), poly(hydroxyl-L -prolyl), and poly(glycyl-glycyl-L -prolyl-glycyl), in aqueous solution, have been studied using ¹³C pulse Fourier transform nmr spectroscopy. From a measurement of the intensities of major and minor resonances in the spectra of the copolypeptides, it was determined that 15–20% of the glycyl-prolyl and glycyl-hydroxyprolyl peptide bonds are cis. Effective rotational correlation times (τ_eff), obtained from measurements of spin-lattice relaxation times (T₁) of individual backbone and side-chain carbons, demonstrated that backbone reorientation is approximately isotropic for the five polypeptides and is characterized by correlation times of ca. 0.3–0.6 nanoseconds as a result of rapid segmental motion. In a given polypeptide glycyl and pyrrolidine residues were found to have the same backbone correlation times, but backbone carbon τ_eff values did decrease as the glycyl content of the peptides increased. A semi-quantitative analysis of C^β, C^γ, and C^δ correlation times suggests that rapid ring motion in both prolyl and hydroxyprolyl involves primarily C^γ and C^β, with the prolyl ring being more mobile than the hydroxyprolyl ring.     

Influence of D and L amino-acid residues on the conformation of peptides in solution: A carbon-13 nuclear magnetic resonance study of cyclo(prolyl-leucyl)

The ¹³C chemical shifts and spin-lattice relaxation times are reported for cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu). The chemical shifts of the D and L leucyl residues in the cyclic peptides differ from each other by 1.8 and 3.6 parts per million for the α and β carbons, respectively. The α-carbons of the prolyl residues differ by 1.0 ppm as a consequence of proximity to a D or an L leucyl residue. The ¹³C spin-lattic relaxation time(T₁) of the prolyl residues, but not the leucyl residues, in both compounds are indicative of difference in conformational equilibria within the pyrrolidine ring in the L -L isomer as compared to the L -D isomer. Anisotropic overall molecular reorientation is not responsible for the differences observed in the T₁ values. The differences in T₁ values and chemical shifts between cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu) appear to result from a difference in conformations of the two diketopiperazine rings.     

Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 2. 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II)

13C spin-lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe. [U-(13)C]ATP and AKe.[U-(13)C]AMP.GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP. (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different (13)C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme-nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e. , they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation-(13)C distances. An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)-(13)C distances to be determined in each of the complexes. Similar measurements of the (31)P relaxation rate made on AKe.ATP and AKe.AMP.GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)-(31)P distances for each adenine nucleotide. These distances, together with the interproton distances determined previously from TRNOESY experiments [Lin, Y., and Nageswara Rao, B. D. (2000) Biochemistry 39, 3636-3646], led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes. These conformations differ significantly from the nucleotide conformations in crystals of AKe. AP(5)A and AKe.AMP.AMPPNP as determined by X-ray crystallography.     

Specific binding of ethanol to cholesterol in organic solvents

Although ethanol has been reported to affect cholesterol homeostasis in biological membranes, the molecular mechanism of action is unknown. Here, nuclear magnetic resonance (NMR) spectroscopic techniques have been used to investigate possible direct interactions between ethanol and cholesterol in various low dielectric solvents (acetone, methanol, isopropanol, DMF, DMSO, chloroform, and CCl(4)). Measurement of (13)C chemical shifts, spin-lattice and multiplet relaxation times, as well as self-diffusion coefficients, indicates that ethanol interacts weakly, yet specifically, with the HC-OH moiety and the two flanking methylenes in the cyclohexanol ring of cholesterol. This interaction is most strong in the least polar-solvent carbon tetrachloride where the ethanol-cholesterol equilibrium dissociation constant is estimated to be 2 x 10(-3) M. (13)C-NMR spin-lattice relaxation studies allow insight into the geometry of this complex, which is best modeled with the methyl group of ethanol sandwiched between the two methylenes in the cyclohexanol ring and the hydroxyl group of ethanol hydrogen bonded to the hydroxyl group of cholesterol.     

Nuclear-magnetic-resonance studies of 5'-ribonucleotide and 5'-deoxyribonucleotide conformations in solution using the lanthanide probe method.

The conformations of the metal-bound 5'-ribonucleotides and 5'-deoxyribonucleotides in aqueous solution at different pH values have been studied using the lanthanide probe method. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from coupling constants, nuclear Over-hauser effects and spin-lattice relaxation times, obtained for the metal-fixed systems. The equilibrium between the two basic conformational combinations for the 5'-nucleotides, anti-(N in equalibrium S)-gg-g'g' and syn-(N in equalibrium S)-gt-g'g' depends on the nature of the furanose ring, the base and also on the state of base protonation and phosphate ionization. The effect of base protonation is particularly strong for the guanine nucleotides.     

The conformation of oxytocin in dimethylsulfoxide as revealed by carbon-13 spin-lattice relaxation times

Information was obtained on rates of overall molecular reorientation and segmental motion of amino acid sidechains of oxytocin in dimethylsulfoxide by determination of spin-lattice relaxation times (T₁) at 25 MHz for carbon-13 in natural abundance in the hormone. The T₁ values of the α-carbons of amino acid residues located in the 20-membered ring of oxytocin are all about 50 msec. The overall correlation time for the hormone backbone was estimated to be 8.8 × 10^?10 sec. The sidechains of Tyr, Ile and Gln undergo segmental motion with respect to the backbone of the ring. The T₁ value of the α-carbon of the Leu residue is greater than for any α-carbon in the ring, indicating an increased mobility of the backbone of the C-terminal acyclic peptide as compared to the ring. The β- and γ-carbons of the Pro residue undergo an exo-endo interconversion with regard to the plane formed by α-carbon, δ-carbon and N atom of the Pro pyrollidine ring. These data are discussed in light of results from other experimental and theoretical studies, including carbon-13 spin-lattice relaxation times for oxytocin in aqueous solution.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1-13C]Tyr-, [15N]Pro- and [1-13C]Val-, [15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-13C]Val-, [15N]Pro signals to the specific residues in bR. The previous assignments of [1-13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C-15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin-lattice relaxation times (T1), spin-spin relaxation times under the condition of CP-MAS (T2), and cross relaxation times (TCH and TNH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin-lattice relaxation times in the rotating flame (1H T1 rho) for the assigned signals were measured in [1-13C]Val-, [15N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid beta-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T1, T2, 1H T1 rho and cross relaxation times. In addition, breakage of the beta-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Structures and populations of conformers of nucleoside monophosphates in aqueous solution. I. General methods of conformation search with lanthanide-ion probes and spin-coupling constants and application to uridine-5′-monophosphate

The Ln(III)-induced shifts and Gd(III)-induced changes of the spin-lattice relaxation times were observed for the ¹H and ¹³C resonances of uridine-5′-monophosphate (5′-UMP) in ²H₂O solutions at pH 1.7–2.0. The vicinal spin-coupling constants changed little by the addition of lanthanide cations, indicating that the conformations of this mononucleotide remained almost the same upon complex formation. A general method was worked out for conformation studies of flexible molecules with lanthanide-ion probes and spin-coupling constants. This method was applied in an extensive computer search for 5′-UMP, and it was concluded that the observed Ln(III)-induced shifts, Gd(III)-induced changes of relaxation times, and vicinal spin-coupling constants could only be accounted for by a mixture of conformers. The populations of the major conformers, gg-3′-endo-anti, and gg-2′-endo-anti, amount to 46 ± 12% and 29 ± 11%, respectively. The correlation between local conformations are discussed on the basis of the present results.     

Intramolecular microdynamical and conformational parameters of peptides from 1H and 13C NMR spin-lattice relaxation. Tetragastrin.

Measurements of 1H and 13C spin-lattice relaxation and 13C nuclear Overhauser enhancement factors are reported for dimethyl sulfoxide solutions of tetragastrin, a pharmacologically active tetrapeptide. The use of the dipolar formalism for predicting 1H and quaternary 13C relaxation rates is discussed. Furthermore, the prospect is opened for the use of quaternary 13C and 1H relaxation times to obtain information on the peptide torsion angles phi, psi, and chi in a way supplementing NMR coupling constant methods presently in use.     

Boat conformations. Analysis and simulation of the complex 1H NMR spectrum of methyl 2,6:3,4-dianhydro-alpha-D-altropyranoside

The complex 1H NMR spectrum of methyl 2,6:3,4-dianhydro-alpha-D-altropyranoside (1) has been analyzed and simulated in detail by using input parameters derived from experimental 1H chemical shifts, long- and short-range coupling constants, spin-lattice relaxation times, and effective, spin-spin relaxation times obtained by trial and error matching of the experimental and simulated spectra. The 13C spin-lattice relaxation times of 1 have also been measured, and along with the 1H-1H long- and short-range coupling constants, have been interpreted in terms of the geometry of 1 defined by molecular dynamics with simulated annealing.     

Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using [U-13C]ATP and [U-13C]ADP

A complete characterization of the conformations of Mn.ADP and Mn.ATP bound to the active site of yeast 3-P-glycerate kinase is presented. These conformations have been deduced on the basis of paramagnetic effects on 13C spin-lattice relaxation rates in [U-13C]nucleotides due to Mn(II), used as a substituent activating cation. The 13C relaxation measurements were performed on exclusively enzyme-bound complexes E.Mn.[U-13C]ATP and E.Mn.[U-13C]ADP at three distinct 13C NMR frequencies: 75.4, 125.7, and 181 MHz. The frequency dependence of the relaxation data has been analyzed in an effort to evaluate distances from the cation for all 10 13C nuclei in the adenosine moieties of E.Mn.ATP and E.Mn.ADP. These distance data, taken along with previously published cation-31P distances, have been used as constraints in the molecular modeling program Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations that are compatible with the distance data. It was possible to model the distances on the basis of a single enzyme-bound conformation for each of the nucleotides. The details of the enzyme-bound Mn.ATP and Mn.ADP conformations are distinguishably different from each other, indicating that structural alterations occur in the enzyme-bound reaction complex as the enzyme turns over. For example, when the adenosine moieties in the bound structures of Mn.ATP and Mn.ADP are superposed, the cation is found to be displaced by approximately 2.4 A between the two conformations, suggesting that these structural changes may involve movements with significant amplitudes. Furthermore, the NMR-determined structures of enzyme-bound Mn.ATP and Mn.ADP are significantly different from those in published X-ray crystal structures of the enzyme-nucleotide complexes.     

NMR spectroscopy, molecular dynamics, and conformation of a synthetic octasaccharide fragment of the O-specific polysaccharide of Shigella dysenteriae type 1

A synthetic octasaccharide fragment (2) of the O-specific polysaccharide (1) of Shigella dysenteriae type 1 has been studied as its methyl glycoside by one- and two-dimensional homo- and heteronuclear NMR spectroscopy. Complete 1H and 13C NMR assignments have been generated, and the 13C spin-lattice relaxation times have been measured for the octasaccharide 2. A congener (6) of this octasaccharide containing one D-galactose residue with a specific 13C label at C-1 has been synthesized and used to measure interglycosidic 13C-1H coupling by the 2D J-resolved 1H NMR method. From the NMR data, three types of conformational restraints were developed: (a) 29 inter-residue, distance restraints; (b) 48 intra-residue, ring atom dihedral angle restraints, and (c) one heteronuclear, inter-residue dihedral angle restraint. The use of these restraints in a restrained molecular dynamics computation with simulated annealing yielded a conformation resembling a short, irregular spiral, with methyl substituents on the exterior.     

Conformation and dynamics of short DNA duplexes: (dC-dG)3 and (dC-dG)4

Natural abundance 13C NMR spectra of duplexed (dC-dG)3 and (dC-dG)4 exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(1) sugar. 4M NaCl causes changes in the 13C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the anti to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

The influence of glycyl residues on the flexibility of peptide hormones in solution. A 13C-nuclear-magnetic-resonance study of luteinizing hormone-releasing hormone (luliberin) and its des-glycinamide10 N-ethylamide analog.

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.     

Solid state NMR and X-ray diffraction studies of alpha-D-galacturonic acid monohydrate

Crystalline alpha-D-galacturonic acid monohydrate has been studied by 13C CPMAS NMR and X-ray crystallography. The molecular dynamics were investigated by evaluating 13C spin-lattice relaxation in the rotating frame (T1rho) and chemical-shift-anisotropy properties of each carbon. Only limited molecular motions can be detected in the low frequency (< 10(4) Hz) range by 13C relaxation time measurements (T1rho) and changes of chemical shift anisotropy properties as a function of temperature. X-ray analysis (at both ambient temperature and 150 K) shows that the acid has the usual chair-shaped, pyranose ring conformation, and that the acid and water molecules are linked, through all their O-H groups, in an extensively hydrogen-bonded lattice.     

Impulse NMR study of the mobility of water adsorbed onto cellulose

An impulse method of nuclear magnetic resonance was used for measuring the times of spin-lattice relaxation in the rotating system of coordinates (RSC) for water molecules adsorbed on cottone cellulose. It has been shown that within the temperature region -10 divided by -40 degrees C the spin-lattice relaxation of water in RSC is conditioned by intermolecular interactions modulated with translation movement. The selfdiffusion coefficient of adsorbed water for the sample with 55% humidity at -10 degrees C is determined as 2.0.10(-9) cm2/s and decreases to 0.3.10(-9) cm2s at -40 degrees C, with activation energy of diffusion equalling 8.1 kcal/mol.     

Conformation of the common purine (beta) ribosides in solution: further evidence for a correlation between N-S state of the ribose moiety and syn-anti equilibrium.

The solution conformations of adenosine, guanosine and inosine in liquid ND3 have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2')-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below - 40 degrees C is the pseudo-rotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T1 measurements on the individual protons it is derived that the N, C(3')-endo, form of the ribose is correlated with an anti conformation of the base (Y approximately 210 degrees to 220 degrees) and the S, C(2')-endo, form of the ribose with a syn conformation of the base (Y approximately 30 degrees to 50 degrees). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.