Search

The mechanical properties of Bombyx mori silk fibers, such as elasticity and tensile strength, change remarkably upon hydration. However, changes in the local conformation and dynamics of individual amino acid residues and change in the dynamics of water molecules due to hydration are not currently well understood on the molecular level. In this work, the conformations and dynamics of the hydrated Bombyx mori silk fibers, including silk cocoon (SC), silk sericin (SS) and silk fibroin (SF), were determined after sustained immersion in water by using

A racemic mixture and two chiral monomers of 2-methyl-1-butyl propiolate, i.e., rac1, R1, and S1, were stereoregularly polymerized with a catalyst, [Rh(norbornadiene)Cl]2, in methanol at 40 °C to obtain the corresponding helical racemic and two chiral polymers, Prac1, PR1, and PS1, and a copolymer, Pco. The 1H and 13C NMR spectra of the racemic and chiral polymers differed, although the NMR spectra of their monomers were the same. The structures of the Pco copolymers with different chiral monomer ratios were analyzed using 1D and 2D NMR, optical rotation, circular dichroism (CD), UV–vis, and computational methods to elucidate the stereochemical effect of the chiral monomers together with the polymerization mechanism. The temperature dependence of 1H and 13C NMR spectra in line shape and intensity indicated that the helical main chain undergoes restricted rotation around the ester methylene bonds −O–CH2– through a three-site jump exchange called an accordion-like helix oscillation (HELIOS). The energetical...

The mechanical properties of Bombyx mori silk fibers, such as elasticity and tensile strength, change remarkably upon hydration. However, changes in the local conformation and dynamics of individual amino acid residues and change in the dynamics of water molecules due to hydration are not currently well understood on the molecular level. In this work, the conformations and dynamics of the hydrated Bombyx mori silk fibers, including silk cocoon (SC), silk sericin (SS) and silk fibroin (SF), were determined after sustained immersion in water by using 13C refocused insensitive nuclei enhanced by polarization transfer (INEPT) NMR, 13C cross-polarization/magic angle spinning (CP/MAS) NMR and 13C dipolar decoupled-magic angle spinning (DD/MAS) NMR. The 13C INEPT NMR spectrum reflects their mobile domain, the 13C CP/MAS NMR spectrum their rigid domain, and the 13C DD/MAS NMR spectrum both domains. The mobile domain of the hydrated SC fiber originates mainly from the hydrated SS part and the rigid domain of the hydrated SC fiber from the hydrated SF part. Moreover, the dynamics of mobile water molecules interacting with the silk fiber was studied by 2H solution NMR relaxation measurements in the silk fiber–2H2O system. Using an inverse Laplace transform algorithm, we were able to identify distinct mobile components in the relaxation times for 2H2O. Our measurements provide new insight relating to the characteristics of the hydrated structure of SC, SS and SF fibers, and the water molecules that interact with them in water. The information is relevant in light of current interest in the design of novel silk-based biomaterials which are usually in contact with blood and other body fluids.

A chiral substituted acetylene, (s)-2-octyl propiolate, was stereoregularly polymerized using a catalyst, [Rh(nbd)Cl]2, at 40 °C in methanol to give the corresponding helical polymer, Ps2OcP. The changes of (1)H and (13)C NMR spectra in line shapes and splitting patterns were consistently interpreted in terms of restricted rotation around the ester O-*C bond, ~O-*C(ε)H(ε)(R)~, R = a branched CH(ε)3 in the ester side chains rather than the helix inversion with the aid of a 3-site jump model. Three peaks due to the branched methyl H(ε) proton and its C(η) carbon observed at 0 °C suggested the formation of three rotamers called A, B, and C, based on the presence of the contracted helix and stretched helix forms that have an intrinsic helical pitch. Furthermore, an accordion-like helix oscillation (HELIOS) along the main chain axis was proposed to explain the temperature dependence spectral changes observed in (1)H and (13)C NMR, UV-vis, and circular dicromism (CD) spectra. The temperature dependence UV-vis and CD spectra of Ps2OcP corroborate the presence of contracted and stretched one-handed helix sense polymers in solution in which the helical pitches and their persistence lengths depend on the temperature.

We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy. The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins. The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and τ values, which are indices of the fluidity of the membrane. These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

Propargyl esters, HC CCH 2 OCOC n H 2n+1 ( 1 : n = 3, 2 : n = 5, 3 : n = 9, and 4 : n = 13), were polymerized using a [Rh(nbd)Cl] 2 catalyst and cocatalyst in an appropriate solvent at 0 °C and 40 °C. Number average molecular weights, M n of the resulting polymers, poly( 1 )–poly( 4 ), were 8000–54,000, and its dispersities ( M w / M n )s and the yields were estimated to be 1.6–3.6 and ca . ∼99%, respectively. These polymers were soluble in CHCl 3 , CH 2 Cl 2 , and THF, and insoluble in CH 3 OH and DMF. The amines, e.g., 2,6-dimethylpyridine and triethylamine worked well as the cocatalysts for the polymerization to give the yields, 93% and 88%, respectively. Further N , N- diethylaniline gave the maximum in cis %, 74%, although pyridine, aniline, pyrrole or imidazole did not work effectively. Remarkable line broadening phenomenon, LBP observed in the 1 H NMR spectra of poly( 1 ) and poly( 2 ) prepared at 0 °C or 40 °C, and poly( 3 ) prepared at 40 °C was correlated with the polymerization temperature and the radical concentration generated in the polymer. The LBP was interpreted by the dipole–dipole interaction between the protons in the polymer and unpaired electrons due to carbon radicals generated by the rotational scission of the cis C C bonds during the polymerization. Their UV–vis spectral shape of poly( 1 )–poly( 4 ) depended on the polymerization temperature and/or the alkyl chain length in the polymer. The XRD powder patterns of poly( 3 ) prepared at 0 °C, and poly( 4 )s prepared at 0 °C and 40 °C showed layer crystal structures with a slightly bent herringbone confirmed by molecular mechanics calculation. The λ max values of poly( 4 ) prepared at 40 °C in methanol showed 317 nm and 340 nm in the solution and on the alumina, respectively.

The poly(propargyl acetate) ( A ) having a helical cis – transoid structure was stereospecifically prepared using the Rh complex catalyst, [Rh(norbornadiene)Cl] 2 , in MeOH or NEt 3 solvent at 0 and 40 °C in moderate yield. Electron spin resonance (ESR) analysis of the polymer revealed the formation of the cis ( B ) and trans ( C ) radicals which were produced through the thermal rotational scission of the helical cis C C bonds in the main-chain during the polymerization. The spatial and geometrical structure was successfully deduced using the two analogues’ polymers in which either methyl or methylene group is deuterated, by the aide of computer simulation of the observed ESR spectra together with the calculation of spin density of the two radicals.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.

13 C NMR Relaxation Study of Segmental Motion of Poly( N e -hydroxyethyl L -glutamine) in Aqueous Solution

Solid-state deuterium NMR ( 2 H NMR) was used to study the dynamics of the tyrosine (Tyr) residue in silk fibroin from Bombyx mori (B. mori) and Samia cynthia ricini (S. c. ricini). Specifically deuterated cocoon silk was obtained by feeding silk worms with Tyr, labeled either at the C β carbon ([3,3- 2 H 2 ]Tyr) or at the aromatic ring ([3',5'- 2 H 2 ]Tyr). The 2 H NMR spectra of the [3,3- 2 H 2 ]Tyr-labeled silk fibroins showed typical rigid powder patterns, indicating that there is essentially no motion about the C α -C β bond axis, both in B. mori and S. c. ricini. In contrast, the 2 H NMR spectra of the [3',5'- 2 H 2 ]-Tyr-labeled silk fibroins consisted of two dynamic components each: a rigid powder contribution, plus a motionally averaged contribution. Hence, some of the Tyr side chains are mobile at the phenolic ring. This motion was characterized to be a π-flip as is typical of aromatic rings. The corresponding 2 H NMR line-shape of the B. mori sample could be successfully simulated by attributing 20% of the signal to a motionally averaged component with a fast rate (10 6 Hz) and the remaining 80% to a much slower component (< 10 3 Hz). Likewise, the simulation of S. c. ricini silk fibroin indicated that 60% of the rings are engaged in fast motional averaging (10 7 Hz), while 40% undergo slow motion (10 4 Hz). Thus, the fraction of the fast component is considerably higher for S. c. ricini silk fibroin than for B. mori, which must be a consequence of their different amino acid sequences. It appears that the side-chain mobility depends on the local packing density around the Tyr residue. We conclude that the Gly-Ala repeats in B. mori silk fibroin are relatively tightly packed. In contrast, a large part of the Gly-rich regions in S. c. ricini are comparatively loosely packed.

Phenyl ring motion in β-form poly( l -phenylalanine) was studied using solid-state deuterium NMR spectroscopy as a function of temperature and echo delay time. The spectra show strong temperature dependence from 173 to 417 K. The line shape is a superposition of two contributions from phenyl groups performing π-flip motion about the C β -C λ axis and fast librations. The π-flipping motion is characterized by a fairly broad distribution of correlation times. The mean correlation times of the distribution vary from 3.8 × 10 −3 to 3.1 × 10 −8 s with increasing temperature of 173 to 338 K, respectively. The apparent activation energy of the π-flip motion is 28 kJ mol −1 , showing low steric hindrance of the rings. There is no evidence of the fast large amplitude motions about the C α -C β axis.

Interactions between Ca2+ and the Asp side chains in the Ca2+-binding site of equine lysozyme were investigated by Fourier-transform infrared (FT-IR) spectroscopy. In the spectrum of equine lysozyme, the intensities of the bands at about 1595 cm-1 and 1578 cm-1 in the region of the COO antisymmetric stretches increased upon Ca2+ binding. In the region of the COO- symmetric stretches, the loss of intensity at about 1388 cm-1 and gains of intensities at about 1423 cm-1 and 1403 cm-1 were observed due to Ca2+ binding to equine lysozyme. The spectral changes for equine lysozyme indicate that the COO- groups of Asp85, Asp90 and Asp91 in the Ca2+-binding site coordinate to Ca2+ in the pseudo-bridging mode, where divalent metal cation is bound to one of the two oxygens in the COO- group and a water molecule is hydrogen bonded to the other oxygen. The results presented here provide further evidence for a high degree of similarity between Ca2+-binding lysozyme and alpha-lactalbumin. The effects of Ca2+ binding on the main-chain conformation of equine lysozyme were compared with those of bovine alpha-lactalbumin and hen egg-white lysozyme.

The characterization of the gellan-paramagnetic metal ion complex has been investigated by ESR and NMR methods in aqueous solution of sodium-type gellan. The resonance of the carboxyl carbon in the d -glucuronate unit in 13C-NMR spectra of gellan is selectively broadened with the addition of the Mn(II) ion in sol state. This is due to the paramagnetic interaction between the carbon and the Mn(II) ion. The resonances of other units of gellan are not affected, showing that Mn(II) ions interact with the carboxyl groups in the d -glucuronate unit. Water proton relaxation measurements indicate that water molecules are well coordinated to the gellan-Mn(II) ion complex in gel state. ESR measurements of the Mn(II) ion give the dissociation constant of 3.2 × 10−5 and 6.5 × 10−4 M, and the number of the binding site of 0.12 and 0.33 per repeated unit in sol and gel states, respectively. The latter suggests that in sol state a Mn(II) ion is surrounded by several residues in a randomly coiled gellan polymer and in gel state a Mn(II) ion binds two or three d -glucuronate units on neighboring gellan molecules.

1 H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to α-lactalbumins from bovine, goat, and human sources. We have identified two 1 H-NMR markers diagnostic of metal ion binding to the high-affinity Ca 2+ -binding site of bovine α-lactalbumin, namely the signals corresponding to the δ-CH 3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca 2+ + bind to this site in either slow (La 3+ , Lu 3+ , Y 3+ , Sr 2+ , Sc 3+ ) or fast (Cd 2+ , Ba 2+ , Pb 2+ ) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y 3+ bind the strongest (Y 3+ > La 3+ , Lu 3+ > Ca 2+ > Sr 2+ > Cd 2+ , Pb 2+ , Ba 2+ > Sc 3+ ). Several metal ions do not alter the 1 1 H spectrum of bovine α-lactalbumin, retaining the protein in an ‘apo-like’ state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co 2+ and Cu 2+ , bind to a second distinct site, termed the ‘zinc site’, and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat α-lactalbumins (bovine and goat α-LA: k off (s − 1 ) ≈ 0.2 to 0.01 from Lu 3+ to Lu 3+ ; human α-LA: k off (s −1 ) ≈ 0.02 to 0.001 from La 3+ to Lu 3+ ). In each case, we found that k off values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human α-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca 2+ off-rate for the bovine protein k off (s −1 ) ≈ 2 to 5), determined using the fluorescent indicator, BAPTA.

The high-affinity Ca2+–binding sites of carp (pI 4.25) and pike (pI 5.0) parvalbumins, as well as those of mammalian calmodulin (CaM) and its C-terminal tryptic half-molecule (TR2C), were analyzed by 207Pb NMR spectroscopy. For the parvalbumins, two 207Pb signals were observed ranging in chemical shift from ≈750 to ≈1260 ppm downfield of aqueous Pb(NO3)2, corresponding to 207Pb2+ bound to the two high-affinity helix-loop-helix Ca2+–binding sites in each of these proteins. Four 207Pb signals, which fall in the same chemical shift window, could be discerned for CaM. Experiments on TR2C permitted the assignment of each signal as due to 207Pb2+ occupying a helix-loop-helix site in either the N- or the C-lobe of the intact protein. 207Pb and 1H NMR titration studies on CaM provided evidence that Pb2+ binding to all four sites occurs simultaneously, in contrast to the behavior of this protein in the presence of Ca2+. Titrations of the 207Pb2+–forms of CaM and TR2C with the antipsychotic drug trifluoperazine demonstrated that drug binding to the exposed hydrophobic surfaces in CaM causes substantial conformational changes and proceeds in a sequential manner – first the C-lobe and subsequently the N-lobe. Finally, the field dependence of CaM-bound 207Pb signals was examined. The 207Pb signal linewidths exhibited a sharp dependence on the square of the external magnetic field, a trend characteristic of relaxation via chemical shift anisotropy. Relaxation studies on TR2C demonstrated that chemical exchange also contributes to the observed linewidths. The large chemical shift dispersion observed for the 207Pb signals of the three proteins studied here illustrates the remarkable sensitivity of this parameter to subtle differences in the chemical environment of the protein-bound 207Pb nucleus. To our knowledge, the data presented in this article comprise the first ever published example of the application of 207Pb NMR spectroscopy to metalloproteins.

The dynamical stacking structure of the phenyl rings in racemic poly(γ-benzyl glutamate) (PBG) deuterated in benzyl methylene or phenyl ring groups was investigated using solid state 2 H NMR quadrupole echo spectroscopy. Line shapes and spin-lattice relaxation times of racemic PBG were compared with those of poly(γ-benzyl l -glutamate) (PBLG). The large amplitude motions along a side chain are rather restricted in the stacked state, while the phenyl rings at the end of the side chain undergo a 180°-flipping motion even in the stacked state as in PBLG. The amplitude of the fast librations in the stacked state is smaller by several degrees than that of PBLG. About 75% of the side chains participate in the stacking for an equi-weight mixture of l - and d -molecules.

The high-affinity calcium-binding sites of bovine and human alpha-lactalbumin as well as equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at delta = -75.9 ppm corresponding to the metal ion bound to the lone Ca(2+)-binding site of the protein. A peak at virtually the identical resonance position (delta = -77.1 ppm) was observed in the analogous experiment with bovine alpha-lactalbumin. In addition, a signal upfield of these (delta = -94.7 ppm) was observed for 113Cd(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca(2+)-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the alpha-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine alpha-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound 113Cd signals for the alpha-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca(2+)-site. Their linewidths also to some extent depend on the concentration of the protein itself.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to investigate side-chain dynamics of poly(γ-benzyl L-glutamate) in the solid state, 2H NMR spectra and spin-lattice relaxation-time T1 of poly(γ-[κ-2H1]benzyl L-glutamate) and poly(γ-[ζ-2H2]benzyl L-glutamate) were measured over a wide temperature range. Line shapes for both samples below room temperature showed static-like axially symmetric powder patterns whose quadrupolar splittings are slightly smaller than the rigid lattice values, and decreased gradually with temperature. This suggests the presence of a rapid, small-amplitude librational motions at both of κ- and ζ-positions. T1 analysis for such librational motion gave the correlation time of the order of 10−11—10−12 s. The spectral intensity started to decrease gradually from 208 K with an increase in temperature, and a remarkable intensity loss was observed at around 323 K. With a further increase in temperature, the spectrum recovered its intensity, and the motional averaged singlet-like spectra were observed for both samples, indicating the presence of rapid, large amplitude motions for the side chains. The T1 minimum was observed at 383 K. T1 and line shapes were fairly well simulated by use of the three-site jump model assuming a Gaussian and a log-Gaussian distributions for the angle between the C–2H bond and the C3 axis and the jump rate, respectively. The average jump rates are located on the relaxation map reported for poly(γ-benzyl L-glutamate). The motional mechanism at higher temperatures follows the WLF type.

To investigate the interaction of Mn(II) ion and gellan gum and carrageenans in the aqueous gels, ESR signal intensity ot Mn(II) ion dissolved in the gels was measured as a function of Mn(II) ion concentration. ESR signals of Mn(II) ion was considerably diminished with a presence of gellan gum and ι-carrageenan. No change was observed for κ-carrageenan. It is suggested that Mn(II) ion strongly interacts with the former two polymers. Analysis of the ESR signal intensity versus Mn(II) ion concentration gave the dissociation constant Kd and the number of binding site n for the Mn(II)—polymer complex. Kd was 0.0012 and 0.14 mol/dm3 for gellan gum and ι-carrageenan respectively, indicating that Mn(II) ion binds gellan gum in the gel state very strongly. The value of n indicated that a Mn(II) ion binds two residues of gellan gum, whereas nearly equimolar binding occurs in ι-carrageenan gel.

Molecular Motion of Poly(acrylate)s Having Mesogenic Side Chains in Deuterated Chloroform Solution Studied by High-Resolution 13 C Nuclear Magnetic Resonance

Side chain dynamics of right-handed α-helical poly(γ-benzyl L -glutamate) (PBLG) was investigated by solid state 2 H NMR. Two main motional modes composed of the large amplitude motions and the rapid and small-amplitude librations along the side chain were extracted from line shapes and T 1 data above the glass-like transition temperature. The motional correlation times and amplitudes of the former are widely distributed, showing the heterogeneity of the side chain region. The phenyl rings at the end of the side chain also undergoes π-flipping. This reorientation is slightly restricted in the racemic complex of PBLG and its enantiomer, left-handed α-helical poly(γ-benzyl D -glutamate), in which phenyl rings from each L and D polymer are considered to stack regularly. The large amplitude motions and the librations are rather restricted in the stack state. About 75% of the side chain participate in the stacking. It is noted that the stacking structure is very flexible on the NMR time scale and the model for the ‘static’ stacks of the phenyl rings should be modified.

Side chain dynamics of right-handed α-helical poly(γ-benzyl L -glutamate) (PBLG) was investigated by solid state 2 H NMR. Two main motional modes composed of the large amplitude motions and the rapid and small-amplitude librations along the side chain were extracted from line shapes and T 1 data above the glass-like transition temperature. The motional correlation times and amplitudes of the former are widely distributed, showing the heterogeneity of the side chain region. The phenyl rings at the end of the side chain also undergoes π-flipping. This reorientation is slightly restricted in the racemic complex of PBLG and its enantiomer, left-handed α-helical poly(γ-benzyl D -glutamate), in which phenyl rings from each L and D polymer are considered to stack regularly. The large amplitude motions and the librations are rather restricted in the stack state. About 75% of the side chain participate in the stacking. It is noted that the stacking structure is very flexible on the NMR time scale and the model for the ‘static’ stacks of the phenyl rings should be modified.

An aliphatic substituted acetylene, n-heptyl propiolate, was stereoregularly polymerised using a catalyst, [Rh(nbd)Cl]2, at 40 °C in methanol to obtain the corresponding helical polymer, PnHepP. The changes in the line shapes and splitting patterns of the 1H NMR spectra were interpreted consistently as representing restricted rotation around the ester O–Ce bond in –O–CeHe2(R)– (R = n-hexyl alkyl chains), rather than helix inversion, with the aid of a 3-site jump model. A two-line peak corresponding to the ester methylene protons of –O–CH2– observed at 30 °C suggested the formation of three rotamers designated as A, B, and C based on the presence of contracted helix and stretched helix forms, each of which has an intrinsic helical pitch with a helical cis–cisoid structure. Furthermore, an accordion-like helix oscillation (HELIOS) along the main chain axis was proposed to explain the temperature dependence of spectral changes observed in the 1H NMR, UV-vis, and IR spectra. The temperature dependence of the UV-vis and 1H NMR spectra of PnHepP corroborated the presence of contracted and stretched one-handed helix sense polymers in solution.

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the segmental motion of poly (L-histidine) (PLH) in aqueous solution, the 13C spin-lattice relaxation time (T 1) was measured at six resonance frequencies (ωC/2π) ranging from 15 to 100 MHz at temperatures from 10 to 80°C. For backbone Cα, plots of log (T 1/ωC) against log (ωC) gave the well-superposed master curve, showing that the time-temperature reduction rule is realized. The shift factor obeyed the Arrhenius-type temperature dependence with the activation energy of 25.0 kJmol-1. Using this activation energy for the temperature dependence of the correlation time, the master curve was well reproduced by the Dejean–Lauprêtre–Monnerie (DLM) model. One of the parameters relating to the segmental motion was τ0/τ1=15, where τ0 and τ1 are the correlation times for the isolated single and correlated pair conformational transitions, respectively. It was found that the spectral density function J(ωC) has the exponent to the correlation time τ1 as ωC J(ωC)∼(ωCτ1)0.71 in the region of ωCτ1≪1.

The interaction of copper with N-phenylphthalamic acid (PPA) cured at various temperatures was investigated by 13C NMR and EPR spectroscopies, as the model system of polyamic acid on copper. EPR spectra prove that copper is dissolved into the PPA–N-methylpyrrolidone solution, producing paramagnetic Cu2+ ions. The 13C NMR resonances of the phthalic group of PPA are selectively broadened due to the paramagnetic interaction between the 13C nuclei and Cu2+ ions, showing binding of Cu2+ to the carboxylate group. Cu2+ ion has no effect on the amide and phenyl groups. Cu2+ ion exchanges rapidly between the carboxylate groups at an exchange rate > 103 s–1 at 23 °C. PPA is imidized to N-phenylphthalimide (PPI) at 150 °C, accompanied by the dissociation of Cu2+. Paramagnetic effects from the Cu2+ ions is not exerted on PPI. The results obtained are compared with the interfacial interaction between polyamic acid and copper.

13C- and water proton-nuclear magnetic relaxation times were measured as functions of temperature and pH to study the interaction of Cu(II) with poly(D-glutamic acid) (PGA) in aqueous solution. In the pH region from 4.5 to 8, the addition of Cu(II) significantly influences the relaxation times of Cγ and Cδ carbons, while those of Cβ, Cα, and peptide C′ are not influenced. The relaxation times of Cγ and Cδ increase with increasing temperature, indicating that a fast exchange between the complexed and uncomplexed states occurs. The scalar relaxation is dominant to line broadening, suggesting that a significant amount of electron spin density is transferred from Cu(II) to Cγ Above pH 9, no paramagnetic effects of Cu(II) upon 13C spectra of PGA were observed. The water proton relaxation of Cu(II) aqueous solution is markedly enhanced by the addition of PGA at an acidic pH, but suppressed at an alkaline pH. These results show that carboxylate groups of PGA and water molecules are bound to Cu(II) at pH≤8, but excluded from Cu(II) at pH≥9.

The large calcium gradient across the plasma membrane creates different environments for intra- and extracellular calcium-binding proteins. The latter are continuously surrounded by 10(-3) M Ca2+, which promotes activation or stabilization of certain proteases, nucleases, or lipases. Other proteins, such as those involved in blood clotting, contain polyelectrolyte regions that are composed of carboxyglutamic or phosphoserine moieties that allow them to interact with Ca2+. In contrast, intracellular calcium-binding proteins, such as calmodulin and troponin C, the trigger proteins for muscle contractions, need to respond to an increase in Ca2+ from 10(-7) to 10(-6) M during cell activation. Evidence is presented that the pairwise arrangement of characteristic helix-loop-helix calcium-binding sites can result in the positive cooperative binding of Ca2+. This can be further promoted by the binding of ligands, drugs, or target proteins Several drug binding sites on calmodulin are allosterically related and their localization on the unusual dumbbell structure of this molecule will be discussed.

Interaction between poly(2-vinylpyridine) (P2VP) and bisacetylacetonato-cobalt(II) (Co(acac)2) in chloroform-d1 (CDCl3) solution was investigated by means of nuclear magnetic resonance spectroscopy. It was found that NMR signal of H-6 proton of the side-chain pyridine ring of P2VP shows a remarkable shift to lower fields by the addition of Co(acac)2. The large shift of H-6 proton indicates that the side-chain nitrogen atom is coordinated to the Co(acac)2. The dependence of the paramagnetic shift on the temperature and Co(acac)2 concentration suggests that the chemical exchange between complexed and uncomplexed states of side-chain of P2VP is rapid. The chemical exchange rate is characterized by an activation energy of 13 kcal/mol. The association constant of 1.2 mol−1 and the intrinsic paramagnetic shift of 9.3 ppm were obtained from the concentration dependence of the paramagnetic shift at room temperature for P2VP—Co(acac)2 complex.

13C NMR parameters such as chemical shift, spin–lattice relaxation time (T1), and nuclear Overhauser enhancement (NOE) were measured for the arginine residue of clupeine in aqueous solution. The correlation time for molecular reorientation of the backbone and side-chain carbons were estimated from T1 and NOE. The correlation time for the α-carbon in the backbone was 0.57 ns at 300 K, indicating rapid segmental motion and that the clupeine molecule is thus flexible. The side chain carbons are more mobile due to the presence of internal motion than the backbone carbon. The internal motion of the side chain of arginine was found to be more restricted compared with that of lysine. This is interpreted in terms of the presence of the bulky guanidino group.

The 400-MHz 1H nuclear magnetic resonance (NMR) studies were carried out on the Ca2+-induced conformational change of calmodulins (CaM's) isolated from scallop testis and pig brain. The resonances were found to be classified approximately into three groups. The resonances of group I, which are perturbed by the binding of Ca2+ to the high-affinity sites, include those of tyrosine-138, epsilon-trimethyllysine-115, histidine-107, tyrosine-99, etc. The previous assignments for tyrosine- (Tyr) 138 [Seamon, K. B. (1980) Biochemistry 19, 207] were corrected. The resonances of group II, which are affected by the binding of Ca2+ to the low-affinity sites, include those of a phenylalanine (Phe), a high field shifted methyl, and a low field shifted alpha-methine. Group III (related to the binding of Ca2+ to both the high-and low-affinity sites) includes the resonances of a Phe, a high field shifted methyl, and threonine-143. It is concluded that sites III and IV are the high-affinity sites. The off-rate of Ca2+ from the high-affinity sites is slower than 50 s-1 while the off-rate from the low-affinity sites is faster than 600 s-1. In the Ca2+-free state, there exists a hydrophobic region containing three phenylalanine (probably Phe-89, Phe-92, and Phe-141), a valine, and an isoleucine in the vicinity of sites III and IV. Tyr-138 is distant from these amino acids. Upon binding of Ca2+ to the high-affinity sites, one of the Phe residues and the valine approach Tyr-138. Similar structural changes were observed between CaM and troponin C when Ca2+ ions are bound to the high-affinity sites. CaM changes in a somewhat different way from troponin C when Ca2+ ions are bound to the low-affinity sites.

The conformational behavior of random copolypeptides of γ-benzyl L-glutamate and γ-methyl L-glutamate was studied by nuclear magnetic resonance in deuterated chloroform—trifluoroacetic acid under various conditions of solvent composition and temperature. The results show that the helix stability of the copolymers is intermediate between those of the two homopolymers, poly(γ-benzyl L-glutamate) and poly (γ-methyl L-glutamate). Broadening and upfield shift are observed for the CH3 proton resonance of the γ-methyl L-glutamate residue of the copolymers. These are attributed to the ring current effect of the phenyl rings on the CH3 protons. The triad sequence of γ-benzyl L-glutamate and γ-methyl L-glutamate in the copolymers was estimated from the CH3 resonance.

The molecular conformation and dynamics of poly(D-glutamic acid) in aqueous solution were studied by 13C nuclear magnetic resonance spectroscopy. Chemical shift, spin—lattice relaxation time (T1), spin—spin relaxation time (T2), and the nuclear Overhauser enhancement (NOE) were measured as functions of pH at 300 K. Increasing pH resulted in upfield shifts of Cα and peptide C′ carbons, reflecting the helix-to-coil transition and downfield shifts of Cβ, Cγ, and Cδ carbons, reflecting the ionization of the side-chain carboxyl group. T1 T2, and NOE increased with increasing pH. The effective reorientational correlation time (τeff) of Cα obtained from the combination of T1 and NOE was 2.8 nsec in the helix region. This indicates that τeff of Cα is determined by not only the overall motion of the molecule but also by appreciable local segmental motions of the backbone. When going to the coil, τeff of Cα decreases by a factor of 4.3 as a result of the onset of rapid segmental motion. There is a progressive increase in T1 values of the side-chain carbons as going away from the backbone, suggesting that the end of the side-chain undergoes more a rapid internal reorientation than the others even in the helix state. The peptide carbonyl carbons relaxes more slowly in D2O than in H2O, suggesting that the relaxation is contributed appreciably from the amide proton which is exchanged with deuterium in D2O solution.

Alkali-chitin was prepared by a simple procedure which included a freezing process in a sodium hydroxide-sodium dodecylsulfate system. Its alkylations were achieved with various alkyl halides of different chain lengths and bulkiness. The affinity toward water or formic acid was remarkably enhanced by the alkylations in spite of the low degree of substitution. The increase in hydrophilicity was considered to be result from the partial destruction of crystalline structure in the chitin molecule. The alkyl-chitin fibers were successfully prepared by spinning the solution of alkyl-chitin in formic acid-dichloroacetic acid mixture into ethyl acetate. The extent of swelling with water was found to correspond both to the chain length and the bulkiness of the alkyl group. The hydroxyl group at the C6 position is considered to be substituted prior to that of C3 position, according to a 13C NMR study. 1H NMR and elemental analysis proved useful in estimting the degree of alkylation even in the case of low degree of substitution.

The proton-magnetic-resonance study has been carried out on poly(γ-benzyl-L-glutamate) (PBLG) film which absorbs dichloromethane (CH2Cl2). Spectra consist of a central sharp doublet superimposed on a broad signal. Separation of the central doublet, which was assigned to adsorbed CH2Cl2, was found to depend on the angle between the film normal and the magnetic field. The angular dependence of the second moment of the doublet was interpreted in terms of an appropriate model for molecular orientation of CH2Cl2 with respect to the PBLG helix and for motion of CH2Cl2.

Escherichia coli thioredoxin (Mr 11,700) usually functions as a hydrogen carrier protein that undergoes reversible oxidation/reduction reactions of its active-site disulfide linkage. By use of a number of assigned and identified resonances in one- and two-dimensional 1H NMR spectra, the two forms of the protein have been compared. Only groups that are relatively close to the active-site Cys-32, Cys-35 linkage such as Trp-28, Trp-31, Phe-27, Ala-29, and Val-25 undergo substantial changes in their 1H NMR chemical shift upon reduction. Various residues that are further removed from the active site, like Tyr-49, Tyr-70, His-6, Phe-12, Phe-81, and Phe-102, appear to be little affected (less than 0.02 ppm) by the reduction, suggesting that the rest of the protein structure is not much affected. Thus, the structural changes that occur upon reduction appear to be localized to the disulfide-containing turn and the central strand of the twisted beta-sheet that directly leads to this turn. Notwithstanding the apparent similarity in the secondary and tertiary structures of the oxidized and reduced forms of the protein, the thermal stability of the protein decreases by 10 degrees C upon the reduction of the single disulfide. This was found by both 1H NMR and near- and far-ultraviolet circular dichroism studies. Oxidized thioredoxin was also more resistant to alkaline denaturation. Furthermore, the exchange rate of the relatively stable slow-exchanging backbone amide protons that are part of the core of the twisted five-stranded beta-sheet of thioredoxin increases substantially after reduction.(ABSTRACT TRUNCATED AT 250 WORDS).