Investigation of metal ions induced conformational changes in N-acetyl alanine methyl ester

The conformation of N-acetyl alanine methyl ester CH₃CONHCH(CH₃)COOCH₃ is determined by CNDO/2 and ab initio calculations with minimal GLO basis sets. The binding sites of small monovalent cations to the ligand are investigated by the ab initio method. The chelate geometry involving peptide and ester carbonyl groups was found to be the most preferential conformation.     

C alpha and C beta carbon-13 chemical shifts in proteins from an empirical database

We have constructed an extensive database of 13C C and C chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical - helix and -sheet geometries for C, and of ca. 2 ppm for C. The side-chain dihedral angle 1 has an effect of up to 0.5 ppm on the C shift, particularly for amino acids with branched side-chains at C. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different , shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and 1 angle, is 0.96 ppm for both C and C. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.     

Unblocked statistical-coil tetrapeptides in aqueous solution: quantum-chemical computation of the carbon-13 NMR chemical shifts

We recently reported a theoretical characterization of representative ensembles of statistical-coil conformations for tetrapeptides with unblocked termini in aqueous solution, at pH 7. The results showed good agreement between the computed Boltzmann-averaged and experimentally-determined values for both the vicinal coupling constants ³J_NH and the -proton chemical shifts. Here, we carry out a cluster analysis of the ensembles of conformations generated in that study, and use them to compute the Boltzmann-averaged values of the quantum-chemical ¹³C chemical shifts for different amino acids in the unblocked tetrapeptides GGXA (where X stands for Phe, Arg, His, Glu, Ile, Lys, Gln, Tyr, Leu, Thr, Ala, Gly and Val). The values of the ¹³C chemical shifts in these thirteen amino acids (for which experimental data are available) were computed by using Density Functional Theory with a 6–311+G(2d,p) basis set. Good agreement is found in terms of both the correlation coefficient (R) and standard deviations of the difference between the computed Bolztmann-averaged and the NMR-determined values for the ¹³C chemical shifts. These results suggest that it may be possible to build a reliable theoretically-derived database of chemical shifts for statistical-coil residues. The results of the current study contribute to our understanding of the relations between chemical shifts, dihedral angles and vicinal coupling constants, ³J_NH. In addition, they can shed light as to how the statistical-coilconformation is related to the conformational preference of more structured states, such as the -helical conformation.     

Oxygen-18 isotope labeling and its effect on carbon-13 chemical shifts of the peptide bond

The effects of 18O isotopes on 13C NMR chemical shifts of peptide carbonyl carbons were found to be 0.028 ppm for glyclyglycine and 0.029 ppm for glycylproline at 50.31 MHz.     

A comparative study of molluscan and crustacean chitin proteoglycans by carbon-13 NMR spectroscopy. Identification of carbohydrate and amino acid contributions and the determination of amino acid chemical shifts in anhydrous formic acid

• 1.1. ¹³C-NMR spectra of formic acid solutions of chitin proteoglycans from cephalopod pen, lamellibranch siphon sheath and crab cuticle have been determined.
• 2.2. Carbohydrate and amino acid components provide well-defined resonances, completely assignable in the case of hexosamine and partially so for protein amino acids.
• 3.3. The individually unique spectra contain information of compositional and chain environment nature.
• 4.4. Spectral data for each protein amino acid, as a formic acid solution, is presented and compared with values for chemical shifts of amino acids and peptides in water.

     

Calcium-induced conformational changes and mutual interactions of troponin components as studied by spin labeling.

The three troponin components, TN-C, TN-I, and TN-T, were spin-labeled with two different derivatives of the nitroxide radical, a maleimide and an imidazole reagent. The ESR spectra of various combinations of labeled and unlabeled components were measured both in the presence and absence of calcium. Conformational changes due to the binding of the components and also due to the binding of calcium were sensitively detected in many combinations as large changes in the spectrum. The conformation of TN-C was modified by both TN-T and TN-I. The effects were larger in the presence of calcium than in its absence. In the presence of calcium, TN-T and TN-I both showed large effects with the maleimide label, while TN-I showed a larger effect than TN-T with the imidazole label. In the absence of calcium, the effect of TN-I was larger than that of TN-T. The senstivitiy of TN-C to calcium was magnified by component binding, since the conformation of TN-C itself was not greatly affected by calcium. The conformation of TN-I was greatly altered only in the presence of both TN-C and calcium. This indicates that the calcium-induced conformational change in TN-C is transmitted to the adjacent TN-I. In reconstituted troponin, the conformation of TN-C was more influenced by TN-I in the presence of calcium and by TN-T in its absence as indicated by the imidazole label. With the maleimide label, TN-I was more influential in the absence of calcium. The effect of calcium on the troponin complex was to make the local environment of the label more rigid. The half-maximal effect was observed at 2 X 10(-6)M calcium with TN-I in various complexes, while it was 10(-5)M with TN-C in the complexes. In any case the calcium effects became discernible at 10(-6)M and saturated at 10(-4)M.     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

Metabolic physiology of alcohol degradation and adaptation in Drosophila larvae as studied by means of carbon-13 nuclear magnetic resonance spectroscopy

Alcohol dehydrogenase-mediated degradation of [2-¹³C]ethanol was followed in third instar larvae of Drosophila by means of ¹³C NMR. The tricarboxylic acid (TCA) cycle intermediates, citrate-C(2),4 and succinate-C2,3; the amino acids, glutamate-C4,3,2, glutamine-C4,3,2, proline-C4, alanine-C2,3 and the carbon nuclei of the glucosyl units of the disaccharide, α,α-trehalose, were intensely labeled in perchloric acid extracts of whole larvae. A model of the intermediary metabolism of ethanol degradation in larvae was formulated from these observations. The C2 atom of ethanol enters the mitochondrial TCA cycle as C2-acetyl-CoA and is converted into the TCA cycle intermediates. The TCA cycle intermediate 2-oxoglutarate(-C4) apparently is readily converted into glutamate(-C4) and subsequently to glutamine(-C4) and proline(-C4). Dietary ethanol is also a substrate for trehalose synthesis. This may occur by an exchange of malate(-C2,3) between its mitochondrial and cytosolic pools. Part of the cytosolic malate(-C2,3) may be diverted into pyruvate then generating alanine(-C2,3) as another product. The other part may be converted into glucose and subsequently into α,α-trehalose by the gluconeogenic pathway. ¹³C natural abundance signals of stored fatty acids and glycerol were readily detectable in chloroform extracts of control larvae. De novo synthesis of fatty acids from labeled ethanol also occurred after a lag period. Our findings show the coordinated nature of metabolic pathways, and we point to its consequences in understanding the dynamics in evolutionary processes.     

Identification of jasmonic acid and its methyl ester as gum-inducing factors in tulips

The purpose of this study was to identify endogenous factors that induce gummosis and to show their role in gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) stems. Using procedures to detect endogenous factors that induce gum in the stem of tulips, jasmonic acid (JA) and methyl jasmonate (JA-Me) were successfully identified using gas–liquid chromatography–mass spectrometry. Total amounts of JA and JA-Me designated as jasmonates in tulip stems were also estimated at about 70–80 ng/g fresh weight, using deuterium-labeled jasmonates as internal standards. The application of JA and JA-Me as lanolin pastes substantially induced gums in tulip stems with ethylene production. The application of ethephon, an ethylene-generating compound, however, induced no gummosis although it slightly affected jasmonate content in tulip stems. These results strongly suggest that JA and JA-Me are endogenous factors that induce gummosis in tulip stems.     

By-products in the analysis of beta-muricholic acid in biological samples as methyl ester triacetate

By-products were formed on analysis of beta-muricholic acid (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholan-24-oic acid) in biological samples by a method involving acid-catalyzed solvolysis of sulfate esters in acetone-methanol, followed by perchloric acid-catalyzed acetylation with acetic anhydride-acetic acid. These products have been identified by mass spectrometry and nuclear magnetic resonance as methyl 3-0,6-0-diacetyl-7-0-(1-methyl-3-oxo-1-butenyl)- and methyl 3-0,7-0-diacetyl-6-0-(1-methyl-3-oxo-1-butenyl)-beta-muricholate, methyl 3-0, 6-0-diacetyl- and methyl 3-0, 7-0-diacetyl-beta-muricholate, and a methyl diacetoxy-cholen-24-oate.     

13C-NMR chemical shifts and the conformations of rigid polypeptides

¹³C-nmr chemical shifts of backbone carbonyl and side-chain β-carbons in polypeptides provide structural information. Recent utilization of substituent effects on ¹³C-nmr chemical shifts (principally γ-effects) has permitted the rationalization of their sequence and conformation dependence when observed in linear, flexible polypeptides. In this report, we apply the γ-effect method to study the ¹³C-nmr chemical shifts observed in solution and in the solid state for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides, which are usually cyclic. As found previously for flexible, linear polypetides, the relative ¹³C-nmr chemical shifts observed for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides are predictable from knowledge of their peptide residue sequence (primary structure) and conformation (secondary structure) via the γ-effect method.     

29Si and 13C chemical shifts and coupling constants of tris(phenyldimethylsilyl)methyl silicon compounds

Silicon-29 (δ ²⁹Si) NMR chemical shifts are reported for the first time for a number of tris(phenyldimethylsilyl)methyl silicon compounds (disilylated derivatives), (PhMe₂Si^A)₃C_αL, where LSi^BR₁R₂R₃, where R varies widely in electronegativity. δ ²⁹Si^B for these series exhibited to some extent good correlations with the electronegativities of the groups bonded to silicon (particularly, δ ²⁹Si^BMe₂X, X H, OH, OCH₃, COCH₃ and Cl). Substitution with electronegative atoms shifts the chemical shift of silicon to high field.The ¹³C NMR spectra of these compounds have been recorded and assigned. The chemical shifts of the α-carbon (C_α) resonances are shown to depend on the type of substituent on the silicon-B, thus ¹³C_α exhibit downfield shifts when X=oxygen ligand. The ¹³C phenyl resonances have been measured and show the same order of o, m and p signals, viz. δ ortho (downfield)>δ para>δ meta.The variation of ²⁹Si-¹H coupling constants with the electronegativity of X was studied.     

Hydrogen-1 and carbon-13 nuclear magnetic resonance conformational studies of the His-Pro peptide bond: conformational behavior of TRH

Cis/trans isomerism of the His-Pro peptide bond provides a convenient model for the effect of a slow conformational change which may have wider biological significance. Above the imidazole pK, His-Pro is conformationally analogous to the (isosteric) peptide Phe-Pro. Protonation of the imidazole sidechain is associated with a large decrease in the cis/trans ratio. Detailed 1H and 13C n.m.r. analysis suggests the importance of electrostatic/hydrogen bonding interactions between the charged imidazolium sidechain and the proline carboxyl as the basis for this effect. In contrast to a previous report, cis/trans isomerism in TRH is shown to be related to titration of the imidazole sidechain, exhibiting a pK of 6.1.     

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.