13C/1H high power double magnetic resonance investigation of collagen backbone motion in fibrils and in solution

${}^{13}\text{C}{}^1\text{H}$ high power double magnetic resonance spectroscopy was used to investigate the mobility of the collagen peptide backbone. [1-¹³C]- and [2-¹³C]-glycine-labeled collagen samples (with >50% enrichment in ¹³C) were prepared via chick calvaria culture. ¹³C n.m.r.² spectra of labeled reconstituted collagen fibrils, of labeled helical collagen in solution, and of unlabeled bovine Achilles tendon collagen were obtained with scalar decoupling and with dipolar decoupling of protons. Proton-enhanced spectra were also obtained using cross-polarization techniques. n.m.r. parameters (linewidths, lineshapes, T₁ values, nuclear Overhauser enhancements, and cross polarization enhancements) were measured for the labeled samples and for collagen in natural abundance. Comparison of ¹³C n.m.r. parameters for bovine Achilles tendon fibrils and for reconstituted chick calvaria collagen fibrils established that chick calvaria collagen is a good model for the molecular dynamics of collagen in vivo.Spin-lattice relaxation times and nuclear Overhauser enhancements for [1-¹³C]- and [2-¹³C]glycine-labeled collagen indicated that R₁ ~2 × 10⁷s^?1 in solution, where R₁ is the diffusion constant for reorientation about the long axis of the molecule. A substantially smaller value for R₁ (2.6 × 10⁶s^?1) was calculated for an axially symmetric ellipsoid of revolution having dimensions appropriate to the collagen helix. The discrepancy between the rigid ellipsoid and n.m.r. values of R₁ suggests that the collagen molecule undergoes torsional reorientation, as well as rod-like reorientation, about its long axis.The T₁ and NOE values measured in the glycine-labeled fibrils show that rapid axial motion (R₁ ~ 10⁷s^?1) persists in the fibrillar state. In the collagen fibril the full width of the glycyl carbonyl powder pattern is 103 p.p.m. This value is substantially smaller than the rigid lattice value, 144 p.p.m., which provides further evidence for motion in the fibril. The observed powder pattern is axially asymmetric, which shows that certain azimuthal orientations are energetically preferred in the fibril. Taken together, the n.m.r. data provide strong evidence that rapid reorientation of the helix backbone occurs in the fibrils. This result shows that formation of a fibrillar structure does not require the existence of a unique set of intermolecular interactions at the helical surfaces.     

Protein dynamics by solid-state nuclear magnetic resonance spectroscopy: Peptide backbone of the coat protein in fd bacteriophage

¹³C and ¹⁵N chemical shift anisotropy and ¹⁵N¹H dipolar powder patterns from backbone sites of the coat protein in fd bacteriophage are not averaged by motion. This means that the polypeptide backbone of the protein has no large amplitude motions rapid compared to 10⁴ Hz. Relaxation studies on the ¹³C_α and ¹⁵N amide resonances indicate the presence of motions on the 10⁹ Hz timescale. These results are reconciled with a model where an otherwise rigid backbone undergoes small amplitude, rapid motions.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Repressor-operator interaction in the lac operon: III. Nuclear magnetic resonance observations with altered amino-terminal DNA binding domains

We have examined the N-terminal 56 amino acid fragment, the domain that can bind DNA independently, from 3-fluorotyrosine-substituted Escherichia coli lac repressor by ¹⁹F-nuclear magnetic resonance. The fragments or “headpieces” from four altered repressers missing each of the tyrosines in turn were examined in parallel. When the wild-type N-terminal fragment is titrated with a 36 base-pair lac operator DNA sequence, the ¹⁹F resonances undergo changes in their chemical shifts that are different from those changes when the N-terminal fragment is titrated with non-specific DNA fragments. By looking at these operator-induced changes as well as pH-dependent effects with all four altered N-terminal fragments, we show systematic correlations with the genetic data. The data lead us to conclude that upon operator DNA binding: (1) tyrosine 7 is displaced to a less polar environment and the higher than normal pK value of the phenolic OH group is decreased; (2) tyrosine 12 does not change much in either its mobility or environment; and (3) tyrosine 17 is involved, as suggested by the genetic data, when the headpiece forms a complex with operator DNA.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C^α and C^β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ_i, ψ_i and χ_i¹, were computed. Secondly, the frequency with which short distances occur between amide, C^α and C^β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein ¹H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor

The assignment of the ¹H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 500 MHz is described. The assignments are based entirely on the known amino acid sequence and the nuclear magnetic resonance data. Individual resonance assignments were obtained for all backbone and C^β protons, with the exception of those of Arg1, Pro2, Pro13 and the amide proton of Gly37. The side-chain resonance assignments are complete, with the exception of Pro2 and Pro13, the N^δ protons of Asn44 and the peripheral protons of the lysine residues and all but two of the arginine residues.     

Hysteretic kinetic behavior of beef liver pyruvate carboxylase.

Natural abundance ¹³C nuclear magnetic resonance (nmr) spectra have been obtained for samples of a variety of native collagens by use of cross-polarization (CP) techniques which permit high resolution natural abundance ¹³C nmr spectra of solids to be obtained with high sensitivity. The CP ¹³C nmr spectra of lyophilized skin and tendon collagens consisted of two broad resonance envelopes spanning a five kHz range. Hydrated tendon collagen gave rise to a CP spectrum very similar to that obtained for the lyophilized sample, indicating that it retains its solid-like properties. In contrast, hydrated skin collagen became denatured under the conditions of the CP experiment and subsequently gave rise to a conventional high-resolution Fourier transform (FT) nmr spectrum. The CP ¹³C nmr spectrum of ivory was similar to those of lyophilized skin and tendon collagens, demonstrating the solid-like character of the collagen in dentine, whereas the CP spectrum of bovine nasal cartilage reflected the presence of highly mobile proteoglycan components in addition to relatively rigid collagen molecules. In the case of ivory, the resolution of the CP spectrum was enhanced by “magic angle” spinning to a degree approaching that of conventional FT ¹³C nmr spectra of denatured collagen in solution. Because of its ability to probe the dynamic properties of solid-like biological molecules, CP ¹³C nmr spectroscopy should be a valuable investigative tool for future studies.     

Investigation of labeled amino acid side-chain motion in collagen using 13C nuclear magnetic resonance

¹³C¹H double magnetic resonance was used to study the interactions and mobility of certain amino acid side-chains of collagen. Samples of collagen, labeled with [3-¹³C]alanine (a small hydrophobic amino acid), [methyl-¹³C]-methionine (a large hydrophobic), [6-¹³C]lysine (positively charged at physiological pH), and [5-¹³C]glutamic acid (negatively charged), were prepared via chick calvaria culture. ¹³C linewidths, lineshapes, NOE² values, and T₁ values were measured for each sample as fibrils and as native (helical) material in solution.The measured T₁ and NOE values for [3-¹³C]alanine-labeled collagen in solution, in conjunction with an ellipsoid model for collagen, indicate that the methyl rotation rate is 2 × 10¹⁰ s^?1 and that the overall rate of diffusion about the long axis is 4× 10⁶ s^?1. These values agree with values for model compounds which undergo internal methyl rotation (Lyerla & Horikawa, 1976) and with previous n.m.r. measurements of the rate of rotational diffusion of backbone ([1-¹³C]- and [2-¹³C]glycine)-labeled collagen (Jelinski & Torchia, 1979). In addition, the n.m.r. data indicate that the terminal carbons of lysine, methionine and glutamic acid in labeled collagen (both in solution and as fibrils) are characterized by reorientation rates of approximately 10⁹ to 10¹⁰ s^?1.Taken together, the n.m.r. data provide strong evidence that the contact regions between the helices in collagen fibrils are fluid and that there is not a unique set of interactions between amino acid side-chains. In this respect, these n.m.r. results support current concepts of globular protein structure which suggest that a variety of conformations, in dynamic equilibrium, are responsible for the structure and function of proteins.     

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time.The three-dimensional structure of yeast tRNA^Phe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.     

Conformation and Orientation of Gramicidin a in Oriented Phospholipid Bilayers Measured by Solid State Carbon-13 NMR

Three analogues of the helical ionophore gramicidin A have been synthesized with ¹³C-labeled carbonyls (¹³C=O) incorporated at either Gly², Ala³, or Val⁷. A fourth compound incorporated ¹³C at both the carbonyl and α-carbon of Gly² within the same molecule. These labels were studied using solid-state, proton-enhanced, ¹³C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180°. The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the ¹³C chemical shift anisotropy (CSA), and in the case of the fourth compound, the ¹³C—¹³C dipolar coupling constant. From the measured CSA and estimates of the orientation of the ¹³C shielding tensor, we are able to determine the direction of the ¹³C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed ππ^6.3_LD single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225-231). The right-handed ππ^6.3_LD single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168-174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and β sheet structures are excluded. In dispersions in which the lipid is in the L_α phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the L_β′ phase the helices are rigid on the timescale of ¹³C-NMR. The configuration of gramicidin A is unaltered by L_α-L_β′ phase transition of the bilayer lipid.     

On the rigidity of RNA in tomato bushy stunt virus

The motional state of RNA in tomato bushy stunt virus, both in the crystalline state and in solution, has been investigated using ³¹P nuclear magnetic resonance methods. It has been found that the RNA is highly immobile in the native virus and it is suggested that the lack of a high-resolution X-ray diffraction pattern for either the RNA or the N-terminal regions of the protein coat molecules (Harrison et al., 1978) is due to static disorder in the crystals. Dynamic disorder has been detected in the virus after treatment with EDTA, which causes a structural change and an increase in particle size.     

Regulatory light chains in myosins.

Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity.Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA.There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. II. Polarization of histidine 57 by substrate analogues and competitive inhibitors

The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin A_δ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to ?15.9, ?16.3 and ?17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of ?18.0 parts per million and the high pH position of ?14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN′ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-l-tryptophan to chymotrypsin A_δ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond.The His-Asp proton resonance was also monitored in complexes of chymotrypsin A_δ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of ?14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH.     

Mobility of elastin chains as determined by 13C nuclear magnetic resonance

Relaxation times and integrated intensities of ¹³C have been obtained from nuclear magnetic resonance spectra of elastin in unstretched calf ligamentum nuchae and indicate that about 80% of the backbone carbonyl carbons have short rotational correlation times, τ_R ～ 40 nanoseconds. τ_R is reduced by only a factor of two when the ligament is in contact with 2 m-KCNS, a strong denaturant. By contrast, the highly ordered chains of collagen in insoluble calf achilles tendon give no spectrum until denatured in 2 m-KCNS, when t_R decreases by many orders of magnitude. These results show that elastin is composed largely of highly mobile chains under physiological conditions, suggesting that configurational entropy has an important role in its elastic properties.     

Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances

The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Glucagon bound to perdeuterated dodecylphosphocholine micelles

The assignment of the ¹H nuclear magnetic resonance spectrum of glucagon bound to perdeuterated dodecylphosphocholine micelles with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 360 MHz is described. Sequential resonance assignments were obtained for all backbone and C^β protons except the N-terminal amino group and the amide proton of Ser2. The assignments of the non-labile amino acid side-chain protons are complete except for the γ-methylene protons of Gln20 and Gln24. These assignments provide a basis for the determination of the three-dimensional structure of lipid-bound glucagon.