Comparison of normal mode analyses on a small globular protein in dihedral angle space and Cartesian coordinate space

Normal mode analyses on the protein, bovine pancreatic trypsin inhibitor, in dihedral angle space and Cartesian coordinate space are compared. In Cartesian coordinate space it is found that modes of frequencies lower than 30 cm(-1) contribute 80% of the total mean-square fluctuation and are represented almost completely by motions in the dihedral angles. Bond angle and length fluctuations dominate in modes above 200 cm(-1), but contribute less than 2% to the total mean-square fluctuation. In the low-frequency modes a good correspondence between patterns of atomic displacements was found, but on average the root-mean-square fluctuations of the Cartesian coordinate modes are 13% greater than their dihedral angle counterparts. The main effect of fluctuations in the bond angles and lengths, therefore, is to allow the dihedral angles to become more flexible. As the important subspaces determined from the two methods overlap considerably, dihedral angle space analysis can be applied to proteins too large for Cartesian coordinate space analysis.     

15N n.m.r. spectroscopy: 36. 1H n.m.r. and 15N n.m.r.spectroscopic investigation on the protonation of polypeptides

¹⁵N n.m.r. (9.12 MHz) spectra of acetamide, polyglycine, poly([l-alanine) and poly(l-leucine) were measured in various acidic solvents. These solvents include dichloroacetic acid (DCA), trifluoroacetic acid (TFA), methane sulphonic acid (MSA) and fluorosulphonic acid (FSA). Full protonation of both amides and polypeptides causes downfield shifts of 17–20 ppm. Furthermore, the concentration dependence of the chemical shift was measured. In solvents which cause partial protonation, decreasing concentration of amide groups may cause downfield shifts up to 8.5 ppm, while in the case of full protonation or in the absence of protonation no concentration dependence is observable. The protonation of peptide groups induces H/D-exchange of the αC proton which was monitored by ¹H n.m.r. spectroscopy. The mechanism of this H/D-exchange is discussed.     

Effects of peptide-binding on the proton n.m.r. spectrum of bovine neurophysin-I

The effects of binding L-phenylalanyl-L-phenylalanine amide and related peptides on the 220 MHz and 300 MHz proton n.m.r. spectra of bovine neurophysin-I were studied. Throughout both the aliphatic and aromatic proton regions, marked binding-induced changes in the protein spectrum occur which are best explained by invoking conformational change within the neurophysin dimer, in addition to direct perturbation of individual protein protons by bound peptide. In the region downfield from 6 p.p.m., a new resonance, centered at 6.45 p.p.m. was resolved in 300 MHz spectra. This resonance is tentatively assigned to a non-exchangeable -NH and undergoes a reversible binding-induced broadening. Also in this region, the binding-induced chemical shift change in the ortho ring protons of Tyr-49 was used to explore additional aspects of the kinetics of peptide-binding. The results indicate that peptides with affinities greater than or equal to 10(4) M-1 exhibit slow to intermediate exchange rates on the time scale of the Tyr-49 chemical shift change, but that fast exchange can be achieved with peptides having affinities approximately equal to 10(2) M-1.     

Reassignment of the methine resonances of D-fructose based on the carbon-13 n.m.r. spectrum of 3-O-methyl-D-fructose

Several disagreements in the ¹³C n.m.r. assignments of the methine carbons of D-fructose exist in the literature. In order to settle these inconsistencies, we examined the ¹³C n.m.r. spectrum of 3-O-methyl-D-fructose. By following the methyl induced shift in this spectrum, as compared to the parent sugar, we identified the alkylated C-3 resonance of all four tautomeric forms of D-fructose. This information, together with our previous identification of the C-5 resonances of the α- and β-forms of D-fructofuranose 6-phosphate, allow the unambiguous identification of all methine carbons of D-fructose in its ¹³C n.m.r. spectrum. The tautomeric composition of 3-O-methyl-D-fructose at 16.5°, in aqueous solution, was found to be as follows: α-pyranose 18%, β-pyranose 37%, α-furanose 11% and β-furanose 34%.     

A computerized approach to the analysis of oligosaccharide structure by high-resolution proton n.m.r.

The 500 MHz proton-n.m.r. spectra of 21 oligosaccharides, predominantly of the lacto-N and lacto-N-neo series and their derivatives containing non-reducing terminal fucose, sialic acid or N-acetylgalactosamine and reduced-end hexitol or hexosaminitol, were examined with 2H2O as solvent. The chemical-shift data obtained from analysis of the spectra were collated with data from other laboratories who have used 250-500 MHz n.m.r. in the analysis of secreted and chemically synthesized oligosaccharides and of the O- and N-linked chains of glycoproteins. A referenced computer library was constructed that includes the chemical shifts of monosaccharides within oligosaccharide sequences that make up the majority of the carbohydrate structures found in mammalian glycoproteins. Examples are given of the computerized interrogation of this library for the assignment of possible structures of unknown oligosaccharides.     

Identification by n.m.r. spectroscopy of a stable intermediate structure in the unfolding of staphylococcal beta-lactamase.

The unfolding of beta-lactamase (penicillinase) from Staphylococcus aureus by guanidinium chloride was followed by using n.m.r. spectroscopy. On the basis of the observation of resonances corresponding to histidine, tyrosine and other amino acid side chains, the existence of a stable partially folded species was demonstrated. These experiments provide detailed characterization of the intermediate that confirms and extends previous characterization by absorption and c.d. spectroscopy and by flow properties. In addition, they show that residues in the N-terminal third of the molecule are affected by the native-to-intermediate transition. Persistent non-equivalence of the two imidazole C2 proton resonances at high guanidinium chloride concentrations is discussed in terms of local sequence effects on the chemical shift.     

Elucidation of the structure and conformation of a methylated tetrasaccharide-alditol acetate by n.m.r. spectroscopy.

The structure of the methylated derivative (1) of the tetrasaccharide-alditol-O-beta-L-rhamnopyranosyl-(1----3)-O-beta-D- xylopyranosyl-(1----4)-O-beta-L-rhamnopyranosyl-(1----2)-1,5-di-O-acetyl -L- arabinitol has been determined solely on the basis of n.m.r. data.     

Conformational study of a somatostatin analogue by high-field n.m.r. spectroscopy, in aqueous solution

The cyclic analogue of somatostatin (SRIF), D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-NH2 (CTC), exhibits good affinity for both opioid and SRIF receptor systems. Its conformational properties were examined in water by high-field proton n.m.r. spectroscopy and compared with results previously obtained with structurally related analogues SMS 201-995 and Sandoz 204-090 in the same solvent. The assignments were made using 2 D-n.m.r. methods, especially long-range connectivities between neighbouring alpha protons, and between beta and aromatic protons. The 3JNH-C alpha H and delta delta/delta T values are compatible with an equilibrium between two gamma turns involving residues 2, 3 and 4 and residues 3, 4, and 5, respectively.     

A 1H n.m.r. study of isotope exchange catalysed by glycolytic enzymes in the human erythrocyte.

The exchange of hydrogen and deuterium atoms between the C-2 position of lactate and solvent was monitored in suspensions of human erythrocytes by using a non-invasive spin-echo p.m.r. method that permits continuous assessment of the rate and the extent of exchange. Exchange rates were measured in cells suspended in buffers made in 2H2O and 1H2O after the addition of L-[2-1H]lactate and L-[2-2H]lactate respectively. The rate of exchange is dependent on the activities of four glycolytic enzymes (fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase and lactate dehydrogenase) and on the concentrations of their substrates. The dependence of the exchange on the following substrates was studied: (1) lactate, (2) the triose phosphates and fructose 1,6-bisphosphate and (3) pyruvate. Observation of the exchange in vitro, in a system produced by mixing the isolated enzymes, permits determination of the individual isotope-exchange equilibrium velocities of the enzymes. The dependence of the equilibrium velocity of human erythrocyte lactate dehydrogenase on NAD+ + NADH concentration was measured. Possible applications of these methods are discussed.     

Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.

A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.     

Selective spin decoupling in the J-resolved two-dimensional 1H n.m.r. spectra of proteins.

Methods were developed where selective homonuclear spin decoupling is used for the identification of the spin systems of individual amino acid residues in J-resolved two-dimensional high field ¹H n.m.r. spectra of proteins. Experiments with the basic pancreatic trypsin inhibitor are shown to illustrate the practical application of these new techniques.     

1H n.m.r. conformational studies on the C-terminal octapeptide of oxyntomodulin, a beta-turn locked by a salt bridge

The octapeptide Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala (Arg4 in the human sequence) is the C-terminal part of porcine oxyntomodulin, an endogeneous peptide which is a potent inhibitor of stimulated acid secretion. This octapeptide exhibits the whole range of biological activities of the parent hormone. In the present work we report an 1H n.m.r. investigation of the conformational properties of the octapeptides of pig and human sequences in dimethylsulfoxide-d6 (DMSO) solution. The various resonances were assigned on the basis of two-dimensional COSY and NOESY experiments. Other experiments such as (i) temperature and concentration dependence of the amide proton chemical shifts, (ii) effects of ionic strength, (iii) comparison of the spectra with different analogues, were performed. We showed that in DMSO, the conformation of the octapeptide is directly related to the ionisation state of the C-terminus carboxyl group of alanine. In carboxylic state, the peptide adopts an extended conformation, while in the carboxylate state the four last residues (Asn-Asn-Ile-Ala) are involved in a type II beta-turn structure probably locked by a salt bridge between the carboxyl group of Ala8 and the epsilon ammonium group of Lys4 (or the guanidinium group of Arg4). These observations provide an insight into the possible conformational tendencies of this peptide in biological media.     

2,3-Dihydroxybenzoate pathway in Pseudomonas putida. 1H n.m.r. study on the ring-cleavage site.

1. Ring cleavage of 2,3-dihydroxybenzoate by cell-free extracts of Pseudomonas putida leads to 2-hydroxy-6-oxo-(2Z,4E)-hexa-2,4-dienoic acid and CO2. 2. The 1H n.m.r. spectrum of the ring-fission product obtained in a 2H2O solution suggests that the extra-diol cleavage occurs between C-3 and C-4.     

Field focusing n.m.r. (FONAR) and the formation of chemical images in man

The first proposals for n.m.r. scanning in medical diagnosis was made by Damadian (1971a; 1972) and were followed by Lauterbur (1973). Damadian's method of scanning used the principle that the forced precessions of a nuclear magnetization under radio frequency (r.f.) driving field specify the conditions for obtaining spatial resolution of the signal producing domains of a nuclear resonance sample. Sufficient coupling of the nuclear spins to the radiation field to produce a signal detectable by r.f. spectroscopy requires that the stringent Bohr frequency condition, hv = microH0/I, be met. It became possible to construct, with the aid of direct current auxiliary coils, a small volume, called the resonance aperture, inside the applied static field of the magnetic resonance experiment. The correct value of H0 for the applied frequency is restricted to this aperture. The technique (Damadian 1972) was developed to provide a method for non-surgically detecting chemical abnormalities in the diseased organs of patients (Damadian 1971a). The first n.m.r. scans of normal patients and of those with malignant disease are discussed.     

The preferred conformations of protected homodito homoheptamethionine peptides. A1 H n.m.r. study in deuterochloroform medium

Detailed analyses of the conformations of the homo-oligopeptide series, Boc-(L-Met)n-OME n = 2--7, in deuterochloroform have been carried out with proton n.m.r. and IR spectroscopy. Well-resolved high field n.m.r. spectra with assignments for the NH and alpha-CH resonances of these homo-methionine peptides are presented. Extensive n.m.r. concentration-dependent chemical shift studies are combined with IR results to delineate the involvement of the various methionine NH protons in intra- and/or intermolecular hydrogen bonding. N.m.r. chemical shift dependencies with temperature and solvent, DMSO-d6, are used to explore the strength of the hydrogen bonds for the various oligopeptides. At low concentrations, where peptide aggregation is absent, the dipeptide is found to be disordered. The tetra- to heptapeptides possess intramolecular hydrogen bonded seven-membered rings at internal residues. The number of internal rings and the oligopeptide self-association increase with increasing peptide chainlength. At intermediate concentrations associations of peptide molecules with folded structures occur with initial association at the C-terminal region. At high concentrations, "in-register" associated extended beta structures are formed.