Conformational studies on poly(N-δ-trimethyl-l-ornithine)

$\text{Poly}\text{(N-δ-}\text{trimethyl-}\text{l}\text{-ornithine}$), (Me₃Orn)_n, is usually not able to attain the α-helical conformation in aqueous solution independent of its pH value; however, it becomes α-helical at low concentrations of sodium perchlorate over a wide pH range according to the circular dichorism (c.d.) spectra. Cl^?, SO₄^2? and H₂PO₄^? do not induce α-helix formation. One can conclude that a distinct topology of the anions bound by the side chains is responsible for the α-helix-inducing effect of some water-structure-breaking anions such as perchlorate. This means that the anions are inserted between the ?N⁺ of the side groups shielding the positive charges repelling one another. The insertion of the anions requires that the water molecules surrounding the ions can be stripped off, which is easily possible if they are water-structure-breaking ones. At higher perchlorate concentrations, the c.d. spectrum changes. It is characterized by a negative shoulder near 208 nm and a pronounced minimum at ≈ 226 nm. With increasing temperature, the c.d. spectrum of the α-helix occurs. Finally the α-helix undergoes a conformational change to the random coil. The apparent transition enthalpy ΔH_vH is remarkably lower than that of the homologue (Me₃Lys)_n, obviously due to a lower cooperativity of the transition. In contrast to poly(l-ornithine), (Orn)_n, the c.d. spectrum of (Me₃Orn)_n remains almost unchanged after adding anionic surfactants such as sodium octyl sulphate (SOS) or sodium dodecyl sulphate (SDS). In organic solvents like methanol or isopropanol, in contrast to (Orn)_a and (Lys)_n, no α-helix formation occurs. However, in mixtures of these alcohols or dioxane with water, α-helix formation is induced by perchlorate, as in pure water. The thermal stability of the α-helix in these systems is increased.     

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.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

13C-nmr chemical shift and conformation of L-alanine residues incorporated into poly(β-benzyl L-aspartate) in the solid state

¹³C-nmr spectra of poly(β-benzyl L-aspartate) containing ¹³C-enriched [3-¹³C]L -alanine residues in the solid state were recorded by the cross polarization–magic angle spinning method, in order to elucidate the conformation-dependent ¹³C chemical shifts of L -alanine residues taking various conformations such as the antiparallel β-sheet, the right-handed α-helix, the left-handed α-helix, and the left-handed ω-helix forms obtained by appropriate treatment. The latter two conformations for L -alanine residues are achieved when L -alanine residues are incorporated into poly(β-benzyl L -aspartate). We found that the alanine C_β carbon show significant ¹³C chemical shift displacement depending on conformational change, and gave the ¹³C chemical shift values at about 17 ppm for the left-handed ω-helix, 14 ppm for the left-handed α-helix, 15.5 ppm for the right-handed α-helix, and 21.0 ppm for the antiparallel β-sheet relative to tetramethylsilane.     

Interactions of D-cellobiose with p-toluenesulfonic acid in aqueous solution: a C NMR study

The effects of adding D₂SO₄, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D₂O were investigated at 23 °C by plotting ¹³C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. ¹³C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D₂SO₄ medium. The C-1 of the α anomer showed a slightly higher response to increasing D⁺ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule.     

Hydrogen-bonding of the ester carbonyls in phosphatidycholine bilayers.

From the behavior of the ¹³C nmr resonances of the carbonyl carbons of phosphatidylcholine vesicles upon the addition of Yb³⁺, Ho³⁺, or Gd³⁺ lanthanide ions it is concluded that of the two peaks the larger downfield one should be assigned to the outside and the smaller upfield one to the inside of the vesicles. The downfield chemical shifts of the α and β carbonyl carbon peaks in vesicles as compared to CCl₄ suggest that the carbonyl oxygens are hydrogen bonded to water in the vesicles. The β-carbonyl oxygen appears more exposed to water in vesicles. Addition of cholesterol to the vesicles produces little chemical shift change suggesting substitution of cholesterol for water in hydrogen bonding to a carbonyl oxygen; the α-carbonyl oxygen is suggested as the more likely acceptor.     

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.     

Pressure dependence of side chain <Superscript>13</Superscript>C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH<Subscript>2</Subscript>

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of ¹³C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH₂ (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B ₁ and B ₂ is dependent on the type of atom and amino acid studied. For H^N, N and C^α the first order pressure coefficient B ₁ is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically ¹³C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.     

NMR study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR chemical shift contour plots

The metastable state silk I structures of Bombyx mori silk fibroin in the solid state were studied on the basis of ¹⁵N- and ¹³C-nmr chemical shifts of Ala, Ser, and Gly residues. The ¹⁵N cross-polarization magic angle spinning (CP/MAS) nmr spectra of the precipitated fraction after chymotrypsin hydrolysis of B. mori silk fibroin with the silk I and silk II forms were measured to determine the ¹⁵N chemical shifts of Gly, Ala, and Ser residues. For comparison, ¹⁵N CP/MAS nmr chemical shifts of Ala were measured for [¹⁵N] Ala Philosamia cynthia ricini silk fibroin with antiparallel β-sheet and α-helix forms. The ¹³C CP/MAS nmr chemical shifts of Ala, Ser, and Gly residues of B. mori silk fibroin with the silk I and silk II forms, as well as ¹³C CP/MAS nmr chemical shifts of Ala residue of P. c. ricini silk fibroin with β-sheet and α-helix forms, are used for the examination of the silk I structure. Both silk I and α-helix peaks are shifted to a lower field than silk II (β-sheet) for the Cα carbons of the Ala residues, while both Cβ carbon peaks are shifted to higher field. However, the silk I peak of the ¹⁵N nucleus of the Ala residue is shifted to lower field than the silk II peak, but the α-helix peak is shifted to high field. Thus, the difference in the structure between the silk I and α-helix is reflected in a different manner between the ¹³C and ¹⁵N chemical shifts. The Cα and Cβ chemical shift contour plots for Ala and Ser residues, and the Cα plot for the Gly residue, were prepared from the Protein Data Bank data obtained for 12 proteins and used for discussing the silk I structure quantitatively from the conformation-dependent chemical shifts. The plots reported by Le and Oldfield for ¹⁵N chemical shifts were also used for the purpose. All these chemical shift data support Fossey's model (Ala: ϕ = −80°, φ = 150°, Gly: ϕ = −150°, φ = 80°) and do not support Lotz and Keith's model (Ala: ϕ = −104.6°, φ = 112.2°, Gly: ϕ = 79.8°, φ = 49.7° or Ala: ϕ = −124.5°, φ = 88.2°, Gly: ϕ = −49.8°, φ = −76.1°) as the silk I structure. © 1997 John Wiley & Sons, Inc.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

13C Nuclear magnetic resonance study of salt induced conformational change of basic polypeptides: Poly (L-lysine), poly (L-arginine), and poly (L-ornithine)

A ¹³C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)_n], poly(L -arginine) [(Arg)_n], and poly (L -ornithine) [(Orn)_n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the C^α and carbonyl carbon signals are observed in the helical state. The upfield shift of the C^β signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)_n and (Arg)_n. However, the displacement of the C^α, C^β, and carbonyl carbons of (Orn)n in the presence of 4M NaClO₄ is found to be almost 50% of that of (Lys)_n and (Arg)_n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T₁), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)_n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)_n, however, the precipitate of the helical polymers is mainly due to aggregation.     

A competing salt-bridge suppresses helix formation by the isolated C-peptide carboxylate of ribonuclease A

The C-peptide of ribonuclease A (residues 1 to 13) is obtained by cyanogen bromide cleavage at Met13, which converts methionine to a mixture of homoserine lactone (giving C-peptide lactone) and homoserine carboxylate (giving C-peptide carboxylate). The helix-forming properties of C-peptide lactone have been reported. The helix is formed intramolecularly in aqueous solution, is stabilized at low temperatures (0 to 20 °C) and also by a pH-dependent interaction between sidechains. The C-peptide lactone helix is about 1000-fold more stable than expected from “host-guest” data for helix formation in synthetic polypeptides.Here we report the failure of C-peptide carboxylate to form an α-helix in comparable conditions. Formation of a salt-bridge between the α-COO^? group and the imidazolium ring of His12⁺ appears to be responsible for the suppression of helix formation. The presence of the Hse13-COO^? … His12⁺ salt-bridge in C-peptide carboxylate is shown by ¹H nuclear magnetic resonance titration of the amide proton resonances of His12 and Hse13, and is expected from model peptide studies. The most probable reason why C-peptide carboxylate does not form an α-helix is that the Hse13-COO^? … His12⁺ salt-bridge competes successfully with a helix stabilizing salt-bridge (Glu9^? … His12⁺).S-peptide (residues 1 to 20 of ribonuclease A) does form an α-helix with properties similar to those of the C-peptide (lactone) helix, which shows that the lactone ring of C-peptide lactone is not needed for helix formation.These results support the hypothesis that a Glu9^? … His12⁺ salt-bridge stabilizes the C-peptide (lactone) helix, and they show that specific interactions between side-chains can be important in preventing as well as in promoting α-helix formation.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.     

Carbon-13 NMR chemical shifts of the common amino acid residues measured in aqueous solutions of the linear tetrapeptides H-Gly-Gly- X-L- Ala-OH

The ¹³C nmr chemical shifts of the common amino acid residues were measured in D₂O solutions of the linear tetrapeptides H-Gly-Gly-X-L -Ala-OH. For Asp, Glu, Lys, Tyr and His, the titration shifts arising from the ionization of te amino acid side chains were also obtained. These data are compared with the corresponding ¹³C chemical shifts in the protected tetrapeptides CF₃CO-Gly-Gly-X-L -Ala-OCH₃, the linear pentapeptides H-Gly-Gly-X-Gly-Gly-OH, and the free amino acids. On this basism the selection of suitable “random coil” ¹³C chemical shifts for conformational studies of polypeptides chain is discussed.     

The effect of ring currents on carbon chemical shifts in cytochromes

Calculations suggest that some carbon chemical shifts in proteinsshould have large ring current shifts (>1 ppm). We present¹³C, ¹⁵N and ¹H assignments forcytochrome c₂ from Rhodospirillum rubrum, compare these withshifts for other cytochromes c, and show that the calculated ring currentshifts are similar to experimentally observed shifts, but that there remainsubstantial conformation-dependent shifts of side-chain carbons. Ringcurrent shifts as large as 6 ppm are observed. We show that the ring currenteffects do not seriously affect the Chemical Shift Index method fordelineating secondary structure, but may have an impact on more precisemethods for generating structural constraints.     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [α-L-Fucp-(1-2)-β-D-Galp-(1-4)-D-Glc, K _a = 4.2 × 10³ M^?1]. The following antigens were bound with a weaker affinity: H-disaccharide α-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Le^y α-L-Fucp-(1-2)-β-D-Galp-(1-4)-[α-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (K _a 1.1?1.7 × 10³ M^?1). The lowest binding was observed for L-fucose (K _a = 2.7 × 10² M^?1) and trisaccharide Le^a, (β-Galp-(1-3)-[α-L-Fucp-(1-4)]-GlcNAc (K _a = 6.4 × 10² M^?1). The Le^d, Le^a, and Le^x pentasaccharides and Le^b hexasaccharide were not bound to LABA.     

13C-nuclear magnetic resonance studies of 85% 13C-enriched amino acids and small peptides: pH effects on the chemical shifts,coupling constants,kinetics of cis-trans isomerisation and conformation aspects

The ¹³C chemical shifts of several 85% ¹³C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH going from 7 to 1 all the δC are shifted more or less upfield with the exception of the carbonyl group of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal.The percentage of cis and trans conformers of glycyl-l-proline and glycyl-l-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-l-proline and only from 77 to 80% for glycyl-l-prolylglycine.The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-l-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the α-protons of the glycine residue, by a lower pK₁ and by a larger ΔδH_α of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-l-proline molecule.The ¹³C-¹³C coupling constants were also studied as a function of pH. The results show that J_Co-Cα of a C-terminal residue, varying from 5 to 6 Hz and reflecting the pK of the carboxylate group, is a linear function of δ_Co and δ_Cα as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the C_α−C_o bond, however, is practically the same in both cases.Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and δ_Co, δ_Cα on the one hand and the J_Co-Cα on the other were established.     

Carbon-13 spin-lattice relaxation studies of intramolecular motion in lysine and a series of oligolysines

¹³C Spin-lattice relaxation times T₁ for individual carbon nuclei have been measured in a series of oligo-l-lysines, as well as lysine and glycine monomers. Anomalous behavior of profiles of T₁ versus pD occurs for lysine and glycine; the T₁ values of the C_α and CO groups are maximal at pH values corresponding to zwitterionic structures. This is interpreted in terms of the hindered intramolecular rotation around the carbonyl-C_α bond at acidic and basic pD values. Lysine monomer manifests a much less pronounced increase in T₁ values from the α- to the ?-carbon than does lysine in an oligomer or polymer. The rate of reorientation of the C_α and C_β carbons of N-terminal groups are faster than those of the central and C-terminal residues, especially at pD greater than 10 for tri-l-lysine hydrochloride and penta-l-lysine acetate. This is interpreted in terms of interaction between the ?-amino groups and the negatively charged carboxyl groups at pD < 10. Segmental motion is shown to make a significant contribution to relaxation of side-chain carbons, making them less sensitive to molecular size than the carbonyl carbons.