13C- and 1H-N.M.R. spectroscopy of permethylated gluco-, galacto-, and manno-pyranoses and their 6-deoxy analogues

The ¹³C- (25.16 MHz) and ¹H-n.m.r (220, 300 MHz) spectra of permethylated mannopyranoses, their 6-deoxy analogues, and permethylated 6-deoxy-gluco- and -galacto-pyranoses have been analysed with the aid of specific trideuteriomethylation, heteronuclear spin-decoupling, and spectrum simulation. Comparison of spectral data for the aldohexose derivatives and their 6-deoxy analogues shows that the ring conformation is not significantly affected by the presence or absence of MeO-6; all compounds are present in the ⁴C₁ (D) or ¹C₄ (L) conformation. Changes in orientation of the MeO groups have distinct effects on the chemical shifts of carbons and protons of the pyranoid rings and of the MeO groups. The possible origins of these effects are discussed.     

Experimental simulation of the environment of the δ opioid receptor. A 500 MHz study of enkephalins in CDCl3

Complexes of [Met⁵] and [Leu⁵]enkephalin amides with 18-crown-6-ether have been studied in CDCl₃ solution by means of 500 MHz NMR spectroscopy, in order to simulate two of the features of the opioid receptor: the apolar environment and the binding of the charged N atom. Contrary to all previus studies in polar solvents the NH resonances are spread in a huge range (ca. 4 ppm) as in the spectra of rigid cyclic peptides. The two observed intramolecular hydrogen bonds are consistent with the existence of a single, folded, conformation, i.e. a C₁₀β-turn in which the Phe⁴ NH is linked to the Tyr¹ CO group.     

Assignment of the 270 MHz nuclear magnetic resonance spectrum of somatostatin in water

The proton nuclear magnetic resonance spectrum of the 14 residue peptide hormone somatostatin in D₂O at 270 MHz has been assigned by comparing the spectra of synthetic analogs with those of the native peptide. Extensive difference double resonance studies of all somatostatins and pH titrations confirmed all assignments. ³J_NHCH values and conventional NMR hydrogen bonding studies confirm the existence of preferred secondary conformations but not with a predominant conformation possessing a β-turn in either of the sequences 7–8–9–10 or 8–9–10–11. More extensive data treatment is needed before the actual conformation(s) of somatostatin is elucidated, but several NMR criteria for conformations are proposed.     

Effects of dilute HCl on yeast tRNAPhe and E. coli tRNAfMet1

HCl treatment of yeast tRNA^Phe under conditions generally used for excision of `Y' base results in structure and conformation changes as monitored by line widths in the PMR spectra at 220 MHz and by optical rotation. Like exposure of E. coli tRNA^fMet₁ causes similar changes in the PMR spectra and optical rotation although no residues are eliminated. Electrophoresis in polyacrylamide gels provides evidence for aggregation in HCl-treated tRNA^fMet₁. One must thus consider a general effect of HCl exposure as well as possible residue removal in assessing induced structural and conformation changes in tRNA.     

A conformational study of the tetrapeptide CH3CO-Ala-Asp-Gly-Lys-NHCH3 corresponding to a β-bend in staphylococcal nuclease

The tetrapeptide sequence Ala-Asp-Gly-Lys occurs as a type I′ β-bend at residues 94–97 in staphylococcal nuclease. We have synthesized theN-acetyl,N′-methylamide derivative of this tetrapeptide and studied its conformation in solution, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. In the synthesis, special attention was paid to the possibility of cyclic aspartimide formation giving rise to mixtures of α- and β-Asp-Gly products. The presence of such a mixture was excluded by infrared, NMR, and other analytical procedures applied to the products and to models for α- and β-linked aspartyl residues. The CD spectra of the protected tetrapeptide in water, methanol, and trifluoroethanol show no evidence of preferred chain conformations. In dimethylsulfoxide-d ₆ , however, the NMR spectra are consistent with the presence of a population of conformers in which the Lys and C-terminal NHCH₃ amide protons are shielded from solvent. Taken together with the observed³J_NH-C ^α _H coupling constants for all residues, this permitted the construction and energetic evaluation of possible conformations in solution. Only one such conformation was fully compatible with the NMR data; this is a type II β-bend in which the Lys and C-terminal NHCH₃ amide protons are close to the Ala C=O group and may form bifurcated hydrogen bonds with it. This conformation can be converted into the conformation existing in staphylococcal nuclease by rotating the plane of the Ala-Asp peptide group by about 120° around a line connecting the Ala and Asp C_α atoms and by making small shifts in dihedral angles elsewhere in the peptide.     

Synthesis and carbon-13 nuclear magnetic resonance spectra of Cyclo(D-leucyl-L-leucine) and Cyclo(L-leucyl-L-leucine)

Cyclo(D -Leu-L -Leu) and cyclo(L -Leu-L -Leu) were synthesized, and their carbon-13 nmr spectra at 65 MHz were examined in dimethylsulfoxide and trifluoroacetic acid solutions. The chemical shift data are consistent with a boat or “twisted” boat conformation of the diketopiperazine ring in both solvents. There was no indication of protonation of the cyclic dipeptides by trifluoroacetic acid. Attempts at polymerizing the cyclic dipeptides were unsuccessful.     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

Phase fluorimetric lifetime spectra. I. In algal cells at 77 K

A new method for decomposing fluorescence emission spectra into their elementary components, based on the simultaneous recording of fluorescence intensity and lifetime vs. the emission wavelength, has been applied to the spectra of algal cells at liquid nitrogen temperature. A model of Gaussian components fits both τ(λ) and F(λ) spectra with the same parameters. The fluorescence lifetimes have been measured by phase fluorimetry at two modulation frequencies: 29 and 139 MHz. The final Gaussian decomposition is able to describe both the 29 and 139 MHz spectra. The following conclusions concerning the fluorescence spectra of Chlorella cells at 77 K can be drawn. These conclusions are also valid with minor changes for the other examined species. (1) An overlapping of different emitting bands occurs in all the spectra; therefore, a direct lifetime reading from phase delay measurement necessitates measurements being made at several frequencies. (2) At the F_max fluorescence level, the lifetime values of the two emissions usually associated with variable fluorescence are 0.53 ns (for B′₁; λ peak 688 nm), and 1.46 ns (for B′₂; λ peak 698 nm); these lifetimes are shorter than those we have measured at room temperature (approx. 1.8 ns). (3) Superimposed on B′₁ and B′₂ and with approximatively the same peak location, two long-lifetime components (B″₁, 4.8 ns; B″₂, 5.6 ns) are present. Two hypotheses can be proposed to explain these emissions: (i) the long-lifetime components arise from subsets of chlorophyll a disconnected from the functional antenna by the cooling process; and (ii) charge recombination in reaction centers leads to delayed fluorescence. (4) In the λ > 710 nm region, two main bands are required to describe the so-called Photosystem I emission: B₃ (0.8 ns; λ peak 715 nm) and B₄ (3.3 ns; λ peak 724 nm). The former band, usually unresolved in the amplitude fluorescence spectra, is a specific finding from lifetime measurements and has been associated with the antenna core of Photosystem I. No additional information has been obtained for B₄. A supplementary small band (B₅, 0.40 ns; $\text{λ}\text{peak}\text{? 740}\text{nm}$) is necessary to take into account the frequency effect and the τ(λ) decrease in the λ > 740 nm spectral range.     

Spectroscopic characterisation of n-octanohydroxamic acid and potassium hydrogen n-octanohydroxamate

The crystal structure and spectroscopic characteristics of n-octanohydroxamic acid and the potassium compound of that acid have been investigated by XRD, XPS, FTIR and Raman spectroscopy. XRD revealed that the acid is in the keto Z conformation with the alkyl chains oriented along the z-direction and hydrogen bonding between hydroxamate moieties. Vibrational spectra confirm this conclusion. Chemical analysis, XRD and XPS established that the potassium compound is the acid salt KH(C₇H₉CONO)₂. The crystal structure showed that the hydroxamate groups are also in the keto Z conformation and this is supported by vibrational spectra. In the acid salt, the two hydroxamate moieties are connected by a symmetrical O-H-O short hydrogen bonded linkage between the two hydroxamate oxygen atoms and this explains the absence of a discernible O-H stretch band in the vibrational spectra. Identification of the vibrational bands displayed is supported by deuteration and ¹⁵N substitution.     

Dielectric behavior of DNA in water–organic co-solvent mixtures

The radiowave dielectric dispersions of DNA in different water–organic co-solvent mixtures have been measured in the frequency range from 100 kHz to 100 MHz, where the polarization mechanism is generally attributed to the confinement of counterions within some specific lengths, either along tangential or perpendicular to the polyion chain. The dielectric dispersions have been analyzed on the basis of two partially different dielectric models, a continuum counterion fluctuation model proposed by Mandel and a discrete charged site model, proposed by Minakata. The influence of the quality of the solvent on the dielectric parameters has been investigated in water–methanol and water–glycerol mixtures at different composition, by varying the permittivity ?_m and the viscosity η of the solvent phase. The analysis of the dielectric spectra in solvents where electrostatic and hydrodynamic interactions vary with the solvent composition suggests that both the two models are able, in principle, to account for the observed high-frequency dielectric behavior. However, while some certain assumptions are necessary about the polyion structure within the Mandel model, no structural prerequisite is needed within the Minakata model, where the polarization mechanism invoked considers a radial counterion exchange with the outer medium, which is largely independent of the local polyion conformation.     

Raman spectra and conformation of the glycerophosphorylcholine headgroup

The Raman and infrared spectra of L-α-glycerophosphylcholine (GPC) and its cadmium complex (GPC·CdCl₂) have been measured for both the anhydrous and hydrate crystals. The Raman spectra of GPC and GPC·CdCl₂ dissolved in water are also reported. All the spectra fall into three distinctive patterns, demonstrating the existence of at least three different kinds of rotational isomers; the first is stable in the anhydrous GPC crystal, the second is stable in the GPC hydrate and GPC·CdCl₂ hydrate crystals and the third is stable in the anhydrous GPC·CdCl₂ crystal and also in aqueous solutions of GPC·CdCl₂. The latter form is reported for the first time.Raman spectra of dimyristoyl-L-α-phosphatidylcholine (L-DMPC) and dipalmitoyl-(L and DL)-α-phosphatidylcholine (L and DL-DPPC) hydrates have been measured below, around and above the transition temperature T₁. The spectra below T₁ indicate that the conformation of the glycerophosphorylcholine (GPC) headgroup in the L-DMPC crystal is similar to that in the L-DPPC crystal. The headgroup conformation in the DL-DPPC crystal is quite different from that in the L-DPPC crystal, although they became the same aftera crystalline trasnformation of DL-DPPC. The changes of the Raman spectra through the T₁ transition for the above crystals show that the headgroup conformation remains unchanged at the beginning o the transition.     

Proton magnetic resonance study of 8-(6-aminohexyl)-aminoadenosine 5′-monophosphate

The nucleotide 8-(6-aminohexyl)-amino adenosine 5′-monophosphate (8-AHA-AMP) has been investigated by 220-MHz proton magnetic resonance spectroscopy. The conformation and ionization state of the nucleotide have been determined. The anti-conformation about the glycosyl bond is the preferred form. The interaction between the hexyldiamino chain and the ribose moiety in this conformation gives rise to unusual ribosyl conformation results. The distribution of conformations about the glycosyl bond has little influence on the effectiveness of this nucleotide analog in the purification of dehydrogenases by affinity chromatography. The chemical shift dependence on pH has been carried out on 8-methylaminoadenosine 5′-monophosphate. The 8-aminoadenine ring is protonated at N1 (pK_α 5.0) and at N7 (pK_α 1.5) in acidic solutions. The protonation at N7 is apparently stabilized by a delocalization of charge onto the 8-amino group. The neutrality of the 8-aminoadenine ring at physiological pH is consistent with the effcient binding of the nucleotide by dehydrogenases. An improved method for the preparation of the 8-AHA-AMP is described.     

Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment

Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health–specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν₁ Raman mode. In addition, analysis of the ν₄ Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy.     

1H-NMR assignments of GM1-oligosaccharide in deuterated water at 500 MHz by two-dimensional spin-echo J-correlated spectroscopy

The ¹H-NMR spectra of the oligosaccharide derived from monosialoganglioside G_M1 (G_M1 = β-d-galactosyl-(1–3)-β-d-N-acetylgalactosaminyl-(1–4)-[α-N-acetylneuraminyl-(2–3)]-β-d-galactosyl-( 1–4)-β-d-glucosylceramide) (G_M1OS) and its reduced form (G_M1OS-R) have been obtained at 500 MHz in D₂O. Through the combined use of one-dimensional and homonuclear two-dimensional spin-echo J-correlated (2D SECSY) spectra of G_M1OS-R, the assignments for the ring protons of G_M1OS are made. Data on chemical shifts and coupling constants of G_M1OS including the α-linked neuraminic acid protons, in aqueous solution, are tabulated. Due to the very small coupling constants (<2 Hz) and the closeness in chemical shifts (<0.04 ppm) for the pair of correlated peaks in the two-dimensional spectrum, the information on the connectivities of the H5 ring protons of the neutral sugar residues is missing. Second-order coupling also blurs this information. Data are compared with those obtained for ganglioside G_M1 in dimethyl sulfoxide (DMSO;the actual composition therein was 97% DMSO-d₆ and 3% D₂O) by T.A.W. Koerner, J. H. Prestegard, P. C. Demou, and R. K. Yu (1983, Biochemistry22, 2676). While the heterogeneity of chemical shifts for the H5, H6a, and H6b protons diminishes in D₂O, that for A-9a and A-9b remains. The latter suggests an intraneuraminic acid conformation involving the glycerol side chain unaffected by the solvent. Moreover, the chemical shifts of the III-1, III-2, and A-4 protons (and perhaps the II-4, IV-2, and A-8 protons) in D₂O exhibit unusual upfield shifts compared with those in DMSO. This indicates that the intramolecular interactions between GalNAc residue III and neuraminic acid present in DMSO are weakened in D₂O. The effect of temperature on the conformation is also examined and appears to be minimal (<0.02 ppm) in the range 22–50 °C.     

Conformational studies on pertrimethylsilyl derivatives of some 6-deoxyaldohexopyranoses and of four less-common aldohexopyranoses by 220-MHz P.M.R. spectroscopy. Substituent and configurational effects on the chemical shifts of the ring protons

The complete interpretation of 220-MHz p.m.r. spectra and the accurate chemical shifts and coupling constants, obtained after computer simulation of the spectra, of the per-O-trimethylsilyl (Me₃Si) derivatives of a number of 6-deoxy-aldohexopyranoses and of β-D-altro-, β-D-allo-, and α- and β-D-talo-pyranose are given. By means of an adapted Karplus equation, the structure of the derivatives has been studied in detail. All of the pyranoid rings occur in the ⁴C₁(D) or ¹C₄(L) chair conformation. The preferred conformation of the C-5—CH₂OSiMe₃ group in the four aldohexopyranoses was found to be dependent on the configuration at C-4. By comparison of Me₃Si-aldohexopyranoses with the corresponding 6-deoxy analogues, it was found that the 6-OSiMe₃ group has no marked effect on the conformation of thering. The influence of this group on the chemical shifts of the ring protons is discussed in terms of electric field and inductive effects. Rules are presented for the estimation of the chemical shifts of the ring protons of Me₃Si-aldohexopyranoses and Me₃Si-6-deoxyaldohexopyranoses.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

A 13C-n.m.r. spectral study of chondroitin sulfates A,B, and C: evidence of heterogeneity

Chondroitin sulfates A, B, and C produce well-resolved ¹³C-n.m.r. spectra which allow for a more complete characterization than that available from their p.m.r. spectra. The ¹³C data fully support earlier evidence as to the main structural features of these glycosaminoglycans, but they also show that many chondroitin preparations are substantially heterogeneous in composition. Thus, spectra of chondroitin A and C have the appearance of composites representative of both types of polymer: specimens of A may contain 25% of the C-type of structural sequence, and C, 30% of the A-type of sequence; 10–20% of unidentified constituents, including a residue bearing a 6-sulfate group, are present in the specimens of chondroitin B. Chemical-shift and ¹J_C-H values found for the L-iduronic acid residues of chondroitin B, as well as the effect of gadolinium nitrate on the relaxation properties of its ¹³C nuclei, indicate that this moiety possesses the α configuration and favors the ¹C₄(L) conformation. Corresponding data for the acetamidodeoxy-D-galactose and D-glucuronic acid residues of the chondroitins are consistent with the β-anomeric configuration and ⁴C₁(D) conformation in all instances.     

Proton NMR study of coordinated imidazoles in low-spin ferric heme complexes. Assignment of single proton histidine resonance in hemoproteins

The proton signals for the coordinated axial imidazoles in a series of low-spin ferric bis-imidazole complexes with natural porphyrin derivatives have been located and assigned. The methyl signals of several methyl-substituted imidazoles have also been resolved for the mixed ligand complexes of imidazole and cyanide ion. The imidazole spectra for the bis complexes are essentially the same as those reported earlier for synthetic porphyrins, with the hyperfine shifts exhibiting comparable contributions from the dipolar and contract interactions. The contact contribution reflects spin transfer into a vacant imidazole π orbital. The spectra of both the mono- and bis-imidazole complex concur in predicting that only the 2-H and 5CH₂ signals of an axial histidine are likely to resonate clearly outside the diamagnetic 0 to ?10 ppm from TMS region in hemoproteins. However, both the 2-H and 4-H imidazole peaks are found to be too broad to detect in a hemoprotein. Hence, it is suggested that the pair of non-heme, single proton resonances in low-spin met-myoglobin cyanides arise from the non-equivalent methylene protons at the 5-position of the histidyl imidazole. Both the resonance positions and relative linewidths in the model compounds are consistent with the data for this pair of protons in myoglobins. The possible interpretations of the average downfield bias of these signals as well as the magnitude of their spacing, are discussed in terms of the conformation of the proximal histidine relative to the heme group.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.