Molecular mobility of polypeptides containing proline as determined by 13C magnetic resonance

The molecular conformations and dynamics of poly(L -prolyl), poly(hydroxyl-L -prolyl), poly(L -prolyl-glycyl), poly(hydroxyl-L -prolyl), and poly(glycyl-glycyl-L -prolyl-glycyl), in aqueous solution, have been studied using ¹³C pulse Fourier transform nmr spectroscopy. From a measurement of the intensities of major and minor resonances in the spectra of the copolypeptides, it was determined that 15–20% of the glycyl-prolyl and glycyl-hydroxyprolyl peptide bonds are cis. Effective rotational correlation times (τ_eff), obtained from measurements of spin-lattice relaxation times (T₁) of individual backbone and side-chain carbons, demonstrated that backbone reorientation is approximately isotropic for the five polypeptides and is characterized by correlation times of ca. 0.3–0.6 nanoseconds as a result of rapid segmental motion. In a given polypeptide glycyl and pyrrolidine residues were found to have the same backbone correlation times, but backbone carbon τ_eff values did decrease as the glycyl content of the peptides increased. A semi-quantitative analysis of C^β, C^γ, and C^δ correlation times suggests that rapid ring motion in both prolyl and hydroxyprolyl involves primarily C^γ and C^β, with the prolyl ring being more mobile than the hydroxyprolyl ring.     

Vibrational averaging of chemical shift anisotropies in model peptides

The effects of chemical shift anisotropy (CSA) are evident in line-shapes or side-band analysis in solid-state NMR, in the observed line positions in partially oriented samples, and in relaxation effects in liquid-state studies. In all of these cases, the effective shielding tensor is influenced by fast vibrational averaging in addition to larger-amplitude internal motions and to overall libration or rotation. Here we compute the contributions of vibrational averaging (including zero-point motions) to the CSA relaxation strengths for the nitrogen and carbonyl carbon in two simple peptide models, and for snapshots taken from a path-integral simulation of a small protein. Because the ¹⁵N shielding tensor is determined by all the atoms of the peptide group, it is less influenced by vibrational motion than (for example) the N–H dipolar interaction, which is more sensitive to the motion of the light hydrogen atom. Computed order parameters for CSA averaging are hence much closer to unity than are N–H dipolar order parameters. This leads to a reduction by about 9% in the magnitude of the amide nitrogen CSA that is needed to fit liquid-state relaxation data. Similar considerations apply to the carbonyl carbon shielding tensor, but in this case the differences between dipolar and CSA averaging are smaller. These considerations will be important for making comparisons between CSA tensors extracted from various NMR experiments, and for comparisons to quantum chemical calculations carried out on static conformers.     

Study of molecular dynamics of bovine carbonic anhydrase B by 13C-NMR in two strongly polarizing magnetic fields. I. Intramolecular mobility of polypeptide chains of the native enzyme

The study of the dynamics of enzyme segmental movement is of considerable importance in the understanding of the physics of the catalytic function of these macromolecules, which cannot be adequately described without introduction of intramolecular mobility of their polypeptide chains. At present high resolution [13C]NMR is mostly used as an effective and selective method for the observation of spectral and relaxation parameters that are sensitive to structure, conformation and local motion. The molecular dynamics of bovine carbonic anhydrase B (carbonate hydrolase EC. 4.2.1.1) in the native form was studied. Measurements of the relaxation parameters (T1, T2 and NOE) of the alpha-carbons of the polypeptide chain in two high magnetic fields (4.7 and 11.7 T) were carried out. The model-free approach of Lipari and Szabo to the interpretation of these experimental data show a satisfactory agreement between theory and experiment for these carbon nuclei if an internal degree of motion such as libration or restricted diffusion in a cone with angular amplitude in the 10 degrees less than theta less than or equal to 20 degrees range and an effective correlation time tau e approximately equal to 6 to 7 x 10(-11) S in addition to the tau R = 3 x 10(-8) S reorientation correlation time of the whole molecular is introduced.     

Fast internal main-chain dynamics of human ubiquitin.

The fast internal dynamics of human ubiquitin have been studied by the analysis of 15N relaxation of backbone amide nitrogens. The amide 15N resonances have been assigned by use of heteronuclear multiple-quantum spectroscopy. Spin lattice relaxation times at 60.8 and 30.4 MHz and the steady-state nuclear Overhauser effect at 60.8 MHz have been determined for 67 amide 15N sites in the protein using two-dimensional spectroscopy. These data have been analyzed in terms of the model free treatment of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The global motion of the protein is shown to be isotropic and is characterized by a correlation time of 4.1 ns rad-1. The generalized order parameters (S2) of backbone amide N-H vectors in the globular region of the protein range from 0.5 to 0.95. No apparent correlation between secondary structure and generalized order parameters is observed. There is, however, a strong correlation between the magnitude of the generalized order parameters of a given N-H vector and the presence of hydrogen bonding of the amide hydrogen or its peptide bond associated carbonyl. Using a chemical shift tensor breadth of 160 ppm, the N-H vectors of peptide linkages participating in one or more hydrogen bonds to the main chain show an average generalized order parameter of 0.80 (SD 0.06), while those amide NH of peptide linkages free of hydrogen-bonding interactions with the main chain show an average order parameter of 0.69 (SD 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrophilicity of polar amino acid side-chains is markedly reduced by flanking peptide bonds

Several amino acid side-chain hydropathy scales have been devised on the basis of solubility and water/organic solvent partitioning data obtained with free amino acids or side-chain analogs. In nearly all cases, these scales are based upon the structure-additivity assumption; it has been assumed that the transfer free energies of the amino acid side-chains are the same in these model compounds as they are in a polypeptide. This assumption is probably wrong. In the present study, deviations from additivity for amino acid side-chains are demonstrated by comparing a theoretically derived scale, which N-acetylamino acid amides. The results show that the flanking peptide bonds dramatically reduce the hydrophilicity of the polar side-chains, with deviations up to several kilocalories (1 kcal = 4.184 kJ) for the charged side-chains at pH 7.0. Further calculation shows that these deviations are due to reductions of 40 to 85% in the unfavorable transfer free energy of the polar functional groups. In addition, proximity of the neighboring amide bonds in the parent molecule (N-acetylglycine amide) decreases the hydrophilicity of the -CONH-backbone unit by 36%. This decrease is expected to be twice as large for -CONH- units in the interior of a polypeptide backbone. The significance of these observations is: (1) valid hydropathy scales can be obtained only with model peptides; (2) deviations from additivity are expected in all solvent systems, including non-polar solvents that are thought to mimic the interior of a membrane; (3) the spontaneous insertion of polypeptides into membranes is likely to occur much more readily than has been previously thought. In order to estimate the free energy of transferring the side-chains and the polypeptide backbone from water to the interior of a lipid bilayer, the results of this study are used to construct a hydropathy scale based upon the partitioning of solutes between water and non-polar solvents. The validity of hydropathy scales that are based on criteria other than solubility and water/organic solvent partitioning data is also discussed.     

Deuterium solid-state NMR investigations of exchange labeled oriented purple membranes at different hydration levels

Oriented purple membranes were equilibrated under controlled (2)H(2)O relative humidity ranging from 15% to 93% and introduced into the magnetic field of an NMR spectrometer with the membrane normal parallel to the magnetic field direction. Deuterium solid-state NMR spectra of these samples resolved four deuteron populations. Deuterons that have exchanged with amide protons of the protein exhibited a broad spectral line shape (<150 kHz). Furthermore, a broadened signal of deuterons tightly associated with protein and lipid is detected at low hydration, as well as two additional water populations that were present when the samples were equilibrated at >/=75% relative humidity. These latter ones are characterized by narrow quadrupolar splittings (<2.5 kHz) and orientation-dependent chemical shifts. Their deuterium relaxation times, measured as a function of temperature, indicate correlation times in the fast regime (10(-10) s) and activation energies of 13 kJ/mol (at 86% relative humidity). Differences in T(1) and T(2) relaxation together with small residual quadrupole splittings show that the mobility of the deuterons is anisotropic. The occurrence of these mobile water populations at high levels of purple membrane hydration (>/=75% relative humidity) correlate with proton pumping activity of bacteriorhodopsin, the fast kinetics of M-decay in the bacteriorhodopsin photocycle, and structural alterations of the protein during the M-state, which have been described previously.     

Topography of the membrane-binding domain of cytochrome b5 in lipids by Fourier-transform infrared spectroscopy

Fourier-transform infrared spectroscopy was used to examine the secondary structure of the membrane-binding domain (nonpolar peptide) of rabbit liver cytochrome b5 in D2O and in the presence of phospholipids and deoxycholate. In all situations, the predominant structure was alpha helix, but an examination of the components of the amide I band in the spectrum of the nonpolar peptide showed that the major peak was shifted from 1655 cm-1 in the lipids to 1650 cm-1 in deoxycholate. This shift to lower frequency, together with a decrease in intensity of the amide II band, is indicative of N-H to N-D exchange of the peptide backbone. A semiquantitative analysis indicated that the alpha helix of the peptide is over 95% exchanged in the presence of deoxycholate but is only 10% exchanged in the presence of lipid. These data suggest that the membrane-inserted portion of the peptide is alpha helical and is largely protected from N-H to N-D exchange by the bilayer. We suggest that this technique appears to provide a general method for determining the type of secondary structure involved in membrane interaction and the percentage of this structure which is involved in the interaction.     

Effect of chain topology on the self-organization and dynamics of block copolypeptides: from diblock copolymers to stars

The effect of chain topology on (i) the peptide secondary structure, (ii) the nanophase self-assembly, and (iii) the local segmental and global peptide relaxations has been studied in a series of model diblock and 3-arm star copolypeptides of poly(epsilon-carbobenzyloxy-L-lysine) (PZLL) and poly(gamma-benzyl-L-glutamate) (PBLG) with PZLL forming the core. Diblock copolypeptides are nanophase separated with PBLG and PZLL domains comprising alpha-helices packed in a hexagonal lattice. Star copolypeptides are only weakly phase separated, comprising PBLG and PZLL alpha-helices in a pseudohexagonal lattice. Phase mixing has profound consequences on the local and global dynamics. The relaxation of the peptide secondary structure speeds up, and the helix persistence length is further reduced in the stars, signifying an increased concentration of helical defects.     

Proton magnetic relaxation in solid poly(L-alanine), poly(L-leucine), poly(L-valine), and polyglycine

Molecular motion in solid poly(L -alanine), Poly(L -leucine), poly(L -valine), and polyglycine has been investigated through measurement of the portion spin-lattice relaxation time at 30 and 60 MHz between 110 and 350°K. Rapid random reoriention of sied-chain methyl groups provides the dominent source of relaxation in the first three; activation energies are 10.5 ± 1 1, 8.5 ± 1 kJ/mol, respectively, significantly lower than in the monomeric crystals. Relaxation times in poyglycine are two orders of magnitude longer than in the monomeric crystals. Relaxation times in polyglycine, significantly lower than in the monomeric crystals. Relaxation times in polyglycine are two orders of magnitude longer and are attributed mainly to segmental motions of the polymer chains. Evidence of nonexponential recovery of nuclear magnetization was encountered in the first three homopolyamino acids but not in polylycine, and was attributed to the correlated time to characterize these motions gave quite good agreement with the data; some improvement was obtained for two polymers using a Cole-Davidson distribution of correlation times. For biopolymers using a Cole-Davidson distribution of correlation times. For biopolymers generally it is concluded that rapid methyl group reorientation is a common dynamical feature and an important source of nuclear magnetic relaxation.     

Effect of calcium on the dynamic behavior of sialylglycerolipids and phospholipids in mixed model membranes. A 2H and 31P NMR study

DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sialic acid headgroup and at the C3 position of the glycerol backbone. The glycolipid was studied as a neat dispersion and in multilamellar dispersions of DMPC (at a concentration of 5-10 mol % relative to phospholipid), using 2H and 31P NMR. The quadrupolar splittings, delta v Q, of the headgroup deuterons were found to differ in the neat and mixed dispersion, suggesting different headgroup orientations in the two systems. In DTSL-DMPC liposomes, two quadrupolar splittings were observed, indicating that the axial and equatorial deuterons make different angles with respect to the axis of motional averaging. The splittings originating from the equatorial and axial deuterons were found to increase and decrease with increasing temperature, respectively, indicating a temperature-dependent change in average headgroup orientation. Longitudinal relaxation times, T1Z, were found to be short (3-6 ms). The field dependence of T1Z suggests that more than one motion governs relaxation. At 30.7 MHz a T1Z minimum was observed at approximately 40 degrees C. At 46.1 MHz the T1Z values were longer and increased with temperature, demonstrating that the dominant rigid-body motions of the headgroup at this field are in the rapid motional regime (greater than 10(8) s-1). DTSL labeled at the glycerol C3 position was studied in DMPC multilamellar dispersions. Whereas two quadrupolar splittings have been observed for other glycolipids labeled at this position, only a single delta nu Q was observed. This shows that the orientation of the C2-C3 segment of DTSL relative to the bilayer normal differs from that of other glycolipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of glycyl residues on the flexibility of peptide hormones in solution. A 13C-nuclear-magnetic-resonance study of luteinizing hormone-releasing hormone (luliberin) and its des-glycinamide10 N-ethylamide analog.

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.     

Amide I band of IR spectrum and structure of collagen and related polypeptides

The infrared amide I band of collagens (rat and cod skin) and related compounds (polyproline, polyglycine, and polytripeptides) was studied. Assignment of amide I-band components for polyproline II and polytripeptides (Gly-Pro-Pro)_n and (Gly-Pro-Gly)_n in the solid state and water solution was made. Three amide I components observed in the polypeptide spectra were attributed to three different peptide CO groups in each triplet. On the basis of this assignment, the interpretation of the amide I multicomponent structure in collagen and isomorphous oligo- and polypeptides was attempted. The ordering of intra- and intermolecular hydrogen bonds involving peptide CO groups in collagen and related compounds was discussed.     

2H nuclear magnetic resonance of exchange-labeled gramicidin in an oriented lyotropic nematic phase

Lyotropic nematic liquid-crystalline phases, such as that formed by potassium laurate/decanol/KCl/water, are found to accept readily large amphiphilic solute molecules. Since these phases spontaneously orient in high magnetic fields, it becomes possible to obtain NMR spectra of biologically interesting solutes in an oriented axially symmetric environment. The amide hydrogens of the peptide backbone of gramicidin D (Dubos) were exchanged for deuterium, and the gramicidin was incorporated into a lyotropic nematic phase made with deuteriated buffer in place of water. 2H NMR spectra of oriented, exchange-labeled gramicidin were then obtained. The strong water signal from the deuteriated buffer was eliminated by using selective excitation and a polynomial subtraction procedure. The 2H NMR spectra at high temperature consist of twelve major quadrupolar doublets. The splittings observed are largely independent of temperature, suggesting a highly rigid backbone structure. Two of the doublets, which are chemically shifted relative to the others, show stronger temperature dependence. These two probably arise from the exchangeable amino hydrogens on the tryptophan indole moieties of the peptide. While we cannot yet assign all of the doublets, the spectra and nuclear magnetic relaxation data are consistent with a rigid slightly distorted beta LD6.3 helix undergoing axially symmetric reorientation about the director of the liquid-crystalline phase. The correlation time for the axially symmetric reorientation is determined by relaxation measurements to be about 10(-7) s.     

Role of water in structural changes of poly(AVGVP) and poly(GVGVP) Studied by FTIR and Raman spectroscopy and ab initio calculations

Two elastin-like poly(pentapeptides), poly(AV1GV2P) and poly(G1V1G2V2P), have been studied in water and in solid state by ATR FTIR and Raman spectroscopy in combination with model ab initio calculations. In aqueous solutions below the transition temperature T(t), a part of the amide groups and of the methyl groups of both polypentapeptides interacts with neighboring water molecules, whereas the other part of amide groups mutually interacts forming a beta-sheetlike structure. Below T(t), poly(AV1GV2P) is dissolved more perfectly, and the water shells around the polymer chains are more closely structured. The suspension of poly(AV1GV2P) formed above T(t) is more compact and, on cooling, resists more to the reverse dissolution, whereas the suspension of poly(G1V1G2V2P) contains more water molecules bound to the carbonyl of amide groups and on backward cooling dissolves fairly reversibly. The measured poly(pentapeptides) tend to form beta-turns due to the conformational transition on the residue between P and V1.     

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.     

Bound water in the collagen-like triple-helical structure.

The ir amide bands of the triple-helical polytripeptides and collagens upon hydration of films are investigated. On the basis of our assignment of the amide I components, the formation of hydrogen bonds between the peptide backbone and structural water is studied. The C1O1--HOH hydrogen bonds are found more ordered than the C3O3--HOH hydrogen bonds. The specific incorporation of water in the triple helix is followed by multistep conformational changes and by increasing of the interpeptide hydrogen-bond strength. The formation of the polypeptide hydrate structure depending on the amino acid composition and the chain length is examined.     

Glycosphingolipids: 2H NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [2,2-2H2]stearic acid, have been studied at a concentration of 10 mol% in bilayers of dimyristoylphosphatidylcholine by 2H NMR. The quadrupolar splitting delta vQ of the C2 deuterons were measured at several temperatures in the range of 30-60 degrees C. Spin-lattice relaxation times T1 of C2 deuterons were determined in the same temperature range for all lipids but globoside. T1 values at 30 and 50 degrees C were unexpectedly short (6-8 ms), indicating reduced mobility of the ceramide acyl chains compared to that of the host phospholipid. At all temperatures, both delta vQ and T1 were essentially identical for the monoglycosylated species, GalCer and GlcCer, indicating that the order and dynamics of the upper portion of the fatty acyl chain are insensitive to this small change in the headgroup structure. In the case of globoside, where the glycolipid headgroup is equivalent to that of GlcCer extended by three sugar residues, values for the quadrupolar splittings associated with the acyl chain C2-position were very close to those obtained for Gal- and GlcCer. In contrast, the delta vQ values obtained for the diglycosyl species, LacCer, were significantly different at all temperatures. This different behavior of LacCer relative to that of the other glycolipids most likely originates from an orientational change of the acyl chain at the C2-position due to the absence of a 4,5 double bond in dihydrosphingosine. T1 values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear magnetic resonance conformational studies on the chemotactic tripeptide formyl-L-methionyl-L-leucyl-L-phenylalanine. A small beta sheet.

Previous work by several groups has shown that the combination of spin--spin coupling constants and spectral density components (derived from spin--lattice relaxation and/or nuclear Overhauser measurements) may aid in the task of conformational determination of peptides in solution. Using the peptide formyl-L-methionyl-L-leucyl-L-phenylalanine, which is a potent specific chemotactic agent for leucocytes, we show the following: (a) that 3JNHCH coupling constants are consistent with a high degree of rigidity in the peptide backbone in solution, (b) that 3H isotopic substitution in combination with relaxation data taken at different Larmor frequencies enables spectral density, and thence conformational, information to be obtained, (c) that side-chain conformations for this molecule mirror, in some aspects, those found in the solid state for other peptides containing the same residues, and (d) that temperature dependence of amide chemical shifts does not have direct implication concerning the existence of intramolecular hydrogen bonds in peptides. We are able to propose a family of conformations which appear to interchange rapidly on the NMR time scale and are characterized by a distribution of side-chain rotamers. The basic backbone conformation is, or closely approximates, a small beta antiparallel pleated sheet and as such suggests a possible mode of receptor--chemotactic peptide interaction.     

Dielectric relaxation studies on bovine ligamentum nuchae

Dielectric relaxation studies of bovine ligamentum nuchae are reported over the frequency range of 1 MHz to 1 GHz and over the temperature range of 23–48°C. A temperature-dependent relaxation process was observed at low megahertz-frequency with the correlation time of around 40 ns. The result is quite similar to that of a synthetic polypentapeptide (VPGVG) and of α-elastin. The relaxation is proposed to arise in part from the peptide libration within the polypentapeptide of bovine ligamentum nuchae.     

Orientational order and dynamics of hydration water in a single crystal of bovine pancreatic trypsin inhibitor.

The orientational order and dynamics of the water molecules in form II crystals of bovine pancreatic trypsin inhibitor (BPTI) are studied by (2)H NMR in the temperature range 6-50 degrees C. From the orientation dependence of the single crystal quadrupole splitting and linewidth, the principal components of the motionally averaged quadrupole interaction tensor and the irreducible linewidth components for the orthorhombic crystal are determined. With the aid of water orientations derived from neutron and x-ray diffraction, it is shown that the NMR data can be accounted for by a small number of highly ordered crystal waters, some of which have residence times in the microsecond range. Most of these specific hydration sites must be located at intermolecular contacts. The surface hydration layer that is also present in dilute solution is likely to be only weakly ordered and would then not contribute significantly to the splitting and linewidth from the protein crystal. To probe water dynamics on shorter time scales, the (2)H longitudinal relaxation dispersion is measured for a polycrystalline BPTI sample. The observed dispersion is dominated by rapidly exchanging deuterons in protein side chains, undergoing restricted rotational motions on a time scale of 10 ns.