A quantitative anharmonic analysis of the amide A band in α‐helical poly(L‐alanine)

Polarized ir spectra of oriented films of α‐helical poly(l ‐alanine) (α‐PLA) have been obtained as a function of residual solvent dichloroacetic acid (DCA). The amide A, B, II, and V regions exhibit multiple bands whose structure depends on the residual DCA content, and those associated with the α_I‐PLA structure have been identified. A calculation of the relevant cubic anharmonic force constants indicates that, contrary to previous assignments, the overtone of amide II(A) is in Fermi resonance with the NH stretch fundamental, whose unperturbed frequency we now find to be at 3314 cm⁻¹, significantly higher than the previously suggested 3279 cm⁻¹. The presence of a structure in addition to the standard α_I‐PLA is indicated by our analysis. © 1999 John Wiley & Sons, Inc. Biopoly 49: 195–207, 1999     

Polarized Raman spectrum of single crystal uridylyl (3′–5′) adenosine: Local Raman tensors of some functional groups

Polarized Raman scattering measurements have been made of a single crystal of uridylyl(3′–5′)adenosine (UpA) by the use of a Raman microscope with 488.0 nm excitation. The UpA crystal belongs to space group P2₁ (monoclinic), and Raman intensities I_aa, I_bb, and I_c′c′, have been determined for each Raman band. These intensities correspond to the aa, bb, and c′c′ components of the crystal Raman tensor, where c′ is defined as an axis perpendicular to the crystallographic a axis in the ac plane. From these experimental data, and by taking the known crystal structure into account, anisotropic and isotropic molecular Raman tensors have been calculated for the following 11 normal modes: ring stretching modes of the adenine residue (protonated) at 1560, 1516, 1330, and 715 cm⁻¹; ring stretching modes of the uracil residue at 1696, 1657, 1615, 1228, and 790 cm⁻¹; PO⁻₂ symmetric stretching mode at 1080 cm⁻¹; P(—)O single bond stretching mode at 801 cm₋₁. These pieces of information of the Raman tensors are considered to be useful for estimating the orientations of the DNA and RNA strands in a biological complex from a polarized Raman spectroscopic measurement of such a complex. © 1998 John Wiley & Sons, Inc. Biopoly 45: 135–147, 1998     

Far-infrared spectra of sequential polypeptides with -helical and polyglycine II structures

The sequential copolymers of glycine and L -alanine, L-valine and L -alanine, L-leucine and L -alanine, and L-phenylalanine and L -alanine and those containing the L-proline residues were synthesized. The infrared spectra in the region from 700 to 200 cm^-1 were measured for these polypeptides with the α-helical conformation or the polyglycine II structure and compared with the spectra of the β-form structures. The results showed that several infrared bands observed in the region from 600 to 200 cm^-1 clearly reflect not only the backbone conformations but also the local conformations of component amino acid residues of polypeptides with the α-helical, β-form and polyglycine II structures.     

Far-infrared spectra of polyalanines with alpha-helical and beta-form structures

Far-infrared spectra of poly-L -alanines having the α-helical conformation and the β-form structure were measured. The spectra of glycine–L -alanine copolymer, silk fibroin, and copoly-D ,L -alanines with different D :L compositions were also measured. In addition to the bands so far reported, four bands at 190, 107, 120, and 90 cm^?1were found for the α-helix conformation and the two bands at 442 and 247 cm^?1 were found for the β form. The 442 cm^?1 band consists of the parallel 432 cm^?1 and perpendicular 445 cm^?1 bands. The 247 cm^?1 band is well defined and has strong dichroism parallel to the direction of stretching. These two bands appear also for silk fibroin and glycine–L -alanine copolymer. All the far-infrared bands of copoly-D ,L -alanines can be interpreted as α-helix bands, the three peaks at 580, 478, and 420 cm^?1 being ascribed to the D -residue incorporated into the right-handed α-helix or to the L -residue in the left-handed α-helix.     

Synthetic α-helical peptides incorporating intercalators for DNA recognition

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz ¹H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential d_NN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an α-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.     

Chemiluminescent assay of β-D-galactosidase based on indole luminescence

We developed a novel chemiluminescent assay of β-D -galactosidase (β-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-β-D -galactopyranoside (X-gal) was used as a substrate for β-gal and also as a light emitter. X-gal was hydrolysed by β-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H₂O₂). H₂O₂ reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of β-gal obtained by this method was 6 × 10⁻¹⁴ mol/L to 6 × 10⁻¹¹ mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using β-gal as the enzyme label. © 1998 John Wiley & Sons, Ltd.     

Spectroscopic studies of pig kidney diamine oxidase-anion complexes

Complexes of pig kidney diamine oxidase with azide, thiocyanate, and cyanide have been characterized by EPR and circular dichroism spectroscopy. Cu(II) d-d bands are observed in the CD spectrum of the resting enzyme at ≈800 nm (12 500 cm⁻¹) and 575 nm (17 400 cm⁻¹). Anion binding produces characteristic changes in the CD spectra. N₃⁻/SCN⁻ → Cu(II) ligand-to-metal charge-transfer transitions are located at 390 nm (25 600 cm⁻¹) and 365 nm (27 400 cm⁻¹), respectively. In addition, an intense new band grew in at ≈500 nm (20 000 cm⁻¹) when azide or thiocyanate were added, which may be assigned as a Cu(II) electronic transition that gains rotational strength in the anion complex. EPR spectra established that the Cu(II)-anion complexes are tetragonal; however, the magnitudes of the anion-induced shifts in the EPR parameters were consistent with substantial perturbations of the Cu(II) electronic ground state in the thiocyanate and cyanide complexes. Prominent superhyperfine splitting was apparent in the EPR spectra of the diamine oxidase complexes with thiocyanate and cyanide. The superhyperfine structure is (at least) partially attributable to endogenous Cu(II) ligands, probably from imidazole.     

Preparation and characterization of oxochromium(IV) and nickel(II) complexes with 5,14-dihydro-7,16-diethyl-(E)- or -(Z)-dipyrido[b,i][1,4,8,11]tetraazacyclotetradecine

The reaction of 5,14-dihydro-7,16-diethyl-(E) or -(Z)-dipyrido[b,i][1,4,8,11]tetraazacyclotetradecine with chromium hexacarbonyl produced the corresponding oxochromium(IV) complexes. These compounds had intense bands in the infrared region at 985 cm⁻¹, which are attributed to the CrO stretching modes. The two complexes gave mass spectra with prominent parent and parent-O peaks. On the other hand the mass spectra for the nickel(II) complexes showed parent and parent-CH₃ peaks. The absorption bands appearing in the energy range greater than 19 000 cm⁻¹ were attributable to the π→π* and CT transitions. Consistent with their diamagnetism, the oxochromium complexes gave well-resolved proton NMR and carbon-13 NMR spectra. The magnitude of downfield shifts for the oxochromium(IV) complex is much larger than that observed for the corresponding nickel(II) complex. This is due to the fact that the positive charge provided by chromium(IV) is greater in magnitude than that by nickel(II).     

Quantum chemical insight on vibration spectra of silica systems

A set of silicate ions and corresponding lithium salts have been quantum chemically (QC) simulated in a “free molecule” approach. The infrared (IR), inelastic neutron scattering (INS), and Raman spectra have been simulated and fitted to the experimentally registered ones. The complete assignment of the vibrational bands along with the intensities and potential energy distribution has been performed. The applicability of the traditionally used quasimolecule Si–O–Si model to the interpretation of bands near 440–480 cm^? 1 and so-called “Boson” peak near 50 cm^? 1 has been critically discussed.     

Conformations of model compounds of proteins. II. Infrared spectra of N-acetyl-amino acid methylamides

N-Acetyl-amino acid methylamides CH₃CONHCHRCONHCH₃ were prepared from L - and DL -alanine, L and DL -α-amino-n-butyric acid, L - and DL -norvaline, DL -norleucine, L - and DL -methionine, L - and DL -leucine, L -aspartic acid and DL -phenylalanine. The deuterium homologs of the type CH₃CONDCHRCONDCH₃, CD₃CONHCHRCONHCH₃, and CH₃CONHCHRCONHCD₃ were also prepared. The infrared spectra of these compounds were measured down to 300 cm^?1 in the crystalline state. The infrared spectra of N-isopropylacetamide CH₃CONHCH(CH₃)₂, N-methylisobutyramide (CH₃)₂CHCONHCH₃ and their deuterium homologs were also measured. The C?O in-plane and out-of-plane bending vibration bands of the CH₃CONHC^α group (amide IVa and VIa) and those of the –C^αCONHCH₃ group (amide IVb and VIb) were assigned from these data. Two crystalline modifications, form I and form II, were found for the compounds prepared from L -alanine, DL -leucine, L -aspartic acid and DL -phenylalanine. The two forms show quite different skeletal vibrations, which suggest, rotational isomerism. Two distinct patterns were found as to the positions of the amide IVa and VIa bands for the above compounds. The two amide bands were found near 630 and 600 cm^?1 in form I, whereas they were found near 600 cm^?1 in form II. The crystals of the remaining compounds were also classified into form I or form II on the basis of the arrangement of the amide bands. The X-ray structure analyses suggest that these two forms have different hydrogen-bond structures.     

Vibrational Raman optical activity of alanyl peptide oligomers: A new perspective on aqueous solution conformation

The vibrational Raman optical activity (ROA) spectra of di- and tri-L -alanine in the range 650–1750 cm^?1 have been measured in H₂O and D₂O solution at high, neutral, and low pH and pD. Corresponding ROA spectra for tetra- and penta-L -alanine have also been obtained, but over a more restricted set of pH and pD conditions. There are similarities with the ROA spectrum of L -alanine below ～ 1200 cm^?1, but the spectra are very different above this wavenumber due to the influence of the vibrational coordinates of the peptide group. The similar overall appearance of the di-, tri-, and tetrapeptide ROA under selected conditions of pH and pD, and of all four peptide ROA spectra in DCl and HCl solutions, in the backbone skeletal stretch region ～ 1050–1200 cm^?1 and the extended amide III region ～ 1250–1350 cm^?1, suggests that the backbone conformation is approximately the same in all four structures. One difference, however, is a shift of a large positive ROA band in H₂O at ～ 1341 cm^?1 in the dipeptide, assigned to C_α–H and in-plane N–H deformations, down to ～ 1331 cm ^?1 in the tripeptide and to ～ 1315 cm^?1 in the tetrapeptide and pentapeptide (the last in HCl due to insufficient solubility in H₂O), which indicates increasing delocalization of the corresponding normal mode with increasing chain length. Our results do not support the suggestion that stabilizing interactions of the zwitterionic end groups in tri-L -alanine at neutral pH leads to a different solution structure to that at high pH. © 1994 John Wiley & Sons, Inc.     

Far-infrared spectra of copoly(- -amino acids)

Far-infrared spectra in the region from 700 to 200 cm^?1 were measured for the copolymers of L -alanine and glycine, those of L -alanine and L -valine, those of L -alanine and L -leucine, and those of L -alanine and L -phenylalanine. The observed spectra were interpreted on the basis of the analysis of the far-infrared spectra of the corresponding homopolymers, and the correlation between the conformations of the copolymers and the kinds of the component amino acids was discussed.     

Preparation and characterization of oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) complexes of 3,3′-(1,2-phenylenediimino)diacrolein

The oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) of 3,3′-(1,2-phenylenediimino)diacrolein were prepared and investigated by means of mass, electronic, vibrational, NMR and ESR spectroscopy as well as magnetic susceptibility measurements. The acetatomanganese(III) and chloroiron(III) complexes were confirmed to be of high spin type. The absorption bands appearing in the energy range greater than 23 000 cm⁻¹ were attributed to π→π^* transitions within a ligand molecule and charge- transfer transitions from metal to ligand. The metal complexes assume the square-planar configuration type since the ligand-field bands were detected in the 12 700–18 500 cm⁻¹ region. Strong bands appearing at 1601 and 1627 cm⁻¹ were assigned to the CC and CO stretching vibrational modes, respectively, and were shifted to lower frequency upon metal-coordination. A VO stretching band was observed at 982 cm⁻¹ for the oxovanadium(IV) complex and a CO stretching band was observed at 1547 cm⁻¹ for the acetatomanganese(III) complex. Upon complex formation the amine proton signal is found to vanish and the aldehydic methine proton signal in the lowest field is shifted upfield for the nickel(II), zinc(II) and palladium(II) complexes. ¹³C NMR spectra support the coordination structure of the complexes which is revealed by ¹H NMR spectra. As judged by the spin Hamiltonian parameters, the oxovanadium(IV) complex is of a square- planar type with an unpaired electron in the d_xy orbital and the copper(II) complex assumes a distorted square-planar coordination due to the presence of five- and six-membered chelate rings with an unpaired electron in the d_x2−y2 orbital.     

Preparation and spectroscopic properties of oxovanadium(IV) and dioxomolybdenum(VI) complexes with tetraaza[14]annulenes containing pyridine rings

The reaction of 5,14-dihydro-7,16-diethyl-(E)- or -(Z)-dipyrido [b, i] [ 1,4,8,11 ] tetraazacyclotetradecine with vanadium(III) chloride led to the corresponding oxovanadium(IV) complexes, while reaction with tetracarbonylbis(piperidine)molybdenum(O) gave the corresponding dioxomolybdenum(VI) complexes. The oxovanadium(IV) complexes showed mass spectra with prominent parent peaks in the El mode. On the other hand the FD mass spectra for the dioxomolybdenum(VI) complexes exhibited the expected parent and parent-O peaks. A VO stretching band was observed at ca. 970 cm⁻¹ for the oxovanadium(IV) complexes. The dioxomolybdenum(VI) complexes had strong bands at 855 and 885 cm⁻¹ in the NaCl region, which are attributable to the MoO symmetric and asymmetric stretching modes. The absorption bands observed above 20 000 cm⁻¹ range were attributed to the CT and π→π^* transitions. Judging from the spin Hamiltonian parameters, the oxovanadium(IV) complexes are of square-planar types with an unpaired electron in the d_xy orbital. The downfield shifts for the dioxomolybdenum(VI) complexes are much larger in magnitude than those observed for the corresponding oxochromium(IV) complexes. This is primarily dependent on the fact that the magnitude of the positive charge fed by molybdenum(VI) is greater than that by chromium(IV).     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998     

Raman spectroscopic analysis of the sodium salt of kappa-carrageenan and related compounds in solution

Laser-Raman spectra of Na⁺ kappa-carrageenan, Na⁺ neocarrabiose 4-sulphate, and neocarrabiose in the region 700–1500 cm⁻¹ are reported for solutions in H₂O and D₂O. The C-1-H-1α vibration, coupled with COH related modes, is assigned to a band at 840 cm⁻¹, close to the maximum of the symmetrical COS stretching (∼850 cm⁻¹). The symmetrical SO stretch is proposed to occur near 1040 cm⁻¹ and is probably coupled with COH vibrations which give rise to strong bands in the region 1000–1100 cm⁻¹. The intense band in the region 730–740 cm⁻¹ is ascribed to a complex ring vibration.     

Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy

Age-mediated deacetylation of hyaluronic acid and dermatan sulfate, and shift of sulfate ester configuration were indicated by infrared spectroscopy. Hyaluronic acid and the three dermatan sulfates (DS₁₈, DS₁₈ and DS₃₅), sequentially precipitated from adult skin with 18%, 28% and 35% ethanol, were analyzed at varying ages. At age 75 years, loss of infrared bands in the 1650-1600 cm⁻¹ region, at 1380 cm⁻¹ and 1320 cm⁻¹ and appearance of a band at 1560 cm⁻¹ were characteristic of hyaluronic acid and DS₃₅,·moreover, in DS₂₈ and DS₃₅ the intensities of the bands at 840 cm⁻¹ and 860 cm⁻ were, respectively, decreased and increased. A low intensity band in the 805-785 cm⁻¹ region was observed in the spectra of DS₁₈ (19–35 years), DS₂₈ (70–80 years) and DS₃₅ (all ages). It intensified in DS₂₈ of the 80-years-olds. In the 75±5-year-old group. ninhydrin-positive material of hyaluronic acid and DS₃₅ increased, while reducing GlcNAc of hyaluronic acid decreased. The data demonstrated hyaluronic acid and DS₃₅ deacetylation and suggested a decrease of equatorial sulfates with infrared band at 840 cm⁻¹ and an incrase of axial sulfates with band at 860 cm⁻¹ in DS₂₈ and DS₃₅ of the 75±5-yearl-old set. Equatorial sulfates with band in the 805±785 cm⁻¹ region apparently decreased in DS₁₈ after 35 years and increased in DS₂₈ of the oldest group.