Protein structure by solid state nuclear magnetic resonance. Residues 40 to 45 of bacteriophage fd coat protein

The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.). Residues 40 to 45 are in a somewhat distorted alpha-helix. This n.m.r. approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field. The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information. These angles are directly related to x, y, z co-ordinates for describing the protein structure.     

N.m.r. imaging of intact biological systems

Internal images of structured objects may be obtained with n.m.r. by labelling component parts with different magnetic field strengths and therefore recognizably different n.m.r. frequencies. A linear field gradient generates a one-dimensional projection of nuclear density and a variety of techniques are employed to manipulate this one-dimensional probe to yield internal images in two and three dimensions. In the past few years, n.m.r. imaging, sometimes also called zeugmatography or spin mapping, has been applied progressively to provide proton images of small phantoms, fruit, vegetables and small animals, and finally to in vivo imaging of the human body; it promises to provide a valuable means of interior investigation of intact biological systems generally. For medical imaging the method is non-invasive, does not use ionizing radiations, appears to be without hazard and penetrates bony cavities without attenuation. Furthermore, other n.m.r. parameters, for example, relaxation times and fluid flow, may also be mapped; there is evidence that the relaxation times from tumours are significantly longer than those from corresponding normal tissue. Effort to date has mostly been concentrated on proton n.m.r., but some work has been done with other nuclei. Three examples are shown of n.m.r. images of intact biological systems: a fruit, an animal and a human system. The discussion includes the quantitative nature of the images, tissue discrimination, the relation between the resolution in the image and image acquisition time, attenuation and phase shift of the r.f. field in the biological tissue, and magnets suitable for n.m.r. imaging. In principle, all conventional n.m.r. techniques can be combined with n.m.r. methods in order to investigate heterogeneous systems. Overhauser imaging is briefly discussed.     

Physical studies on phosphonium phosphatidylcholine. A unique [31P]phosphorus nuclear-magnetic-resonance probe for model and biological membranes.

1. Distearoyl phosphatidylcholine and the phosphonium analogue, in which the nitrogen atom is replaced by phosphorus, show similar gel-liquid crystalline transition temperatures as detected by differential scanning calorimetry. 2. The temperature-dependence of the 31P n.m.r. (nuclear-magnetic-resonance) linewidths of the phosphate resonances of sonicated vesicles of distearoyl phosphatidylcholine and the phosphonium analogue are similar. Below the phase-transition temperature the linewidths decrease as the temperature is raised. Above the phase-transition temperature the phosphate resonances are relatively temperature-independent. The phosphonium 31P n.m.r. signal exhibits the same pattern of temperature-dependence. 3. The 31P n.m.r. phosphonium resonance is sensitive to the paramagnetic shift reagent, K3Fe(CN)6. Use of K3Fe(CN)6, together with Nd(NO3)3, enabled the determination of the trans-bilayer distribution of egg-yolk phosphatidylcholine and its phosphonium analogue in co-sonicated vesicles. Both are distributed comparably across the bilayer of the vesicles. 4. The phosphonium 31P n.m.r. signal is much sharper than the corresponding phosphate resonance in both sonicated and unsonicated dispersions of the phosphatidylcholine analogue. 5. The properties of the phosphonium analogue of phosphatidylcholine are discussed in terms of its suitability as a probe of membrane structure.     

Observations on the phosphate status and intracellular pH of intact cells, protoplasts and chloroplasts from photosynthetic tissue using phosphorus-31 nuclear magnetic resonance.

Individual pools of intracellular inorganic phosphate (Pi) can be observed in the dark in intact cells, protoplasts and chloroplasts from photosynthetic tissue by using 31P nuclear magnetic resonance (n.m.r.). Estimates for the pH of vacuolar and extravacuolar compartments are reported although it is shown that intracellular pH is determined by the pH of the suspending medium. Mannose treatment of asparagus (Asparagus officinalis) cells and spinach (Spinacia oleracea) protoplasts results in the inhibition of photosynthesis. The mechanism of mannose phosphate sequestration of free Pi is supported by the 31P n.m.r. spectra of mannose-treated tissue. There is a fundamental difference in 31 P n.m.r. spectra of mannose-treated spinach protoplasts and asparagus cells, reflecting a difference in the availability of vacuolar Pi for cellular metabolism in these species. The 31P n.m.r. spectrum of intact spinach chloroplasts is reported.     

Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance.

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.     

Prediction of protein folding pathways.

Recent 1H nuclear magnetic resonance (n.m.r.) hydrogen exchange experiments on five different proteins have delineated the secondary structures formed in trapped, partially folded intermediates. The early forming structural elements are identifiable through a technique described in this work to predict folding pathways. The method assumes that the sequential selection of structural fragments such as alpha-helices and beta-strands involved in the folding process is founded upon the maximal burial of solvent accessible surface from both the formation of internal structure and substructure association. The substructural elements were defined objectively by major changes in main-chain direction. The predicted folding pathways are in complete correspondence with the n.m.r. results in that the formed structural fragments found in the folding intermediates are those predicted earliest in the pathways. The technique was also applied to proteins of known tertiary structure and with fold similar to one of the five proteins examined by 1H n.m.r. The pathways for these structures also showed general consistency with the n.m.r. observations, suggesting conservation of a secondary structural framework or molten globule about which folding nucleates and proceeds.     

Termini of Salmonella flagellin are disordered and become organized upon polymerization into flagellar filament

The terminal regions of Salmonella flagellin are essential for polymerization to form the flagellar filament. It has recently been suggested, on the basis of results from circular dichroism spectroscopy and scanning calorimetry, that these regions are disordered in solution. We report here direct evidence for disorder and mobility in the terminal regions of flagellin using 400 MHz proton nuclear magnetic resonance (n.m.r.) spectroscopy. Comparison of the n.m.r. spectra of monomeric and polymeric flagellin shows that the terminal regions become organized when polymerized to form the filament.     

[19F]fluorine nuclear-magnetic-resonance study of the interaction of difluoro-oxaloacetate with aspartate transaminase.

Difluoro-oxaloacetate interacts with the aldimine form of aspartate transaminase to give a complex, the dissociation constant of which has been determined spectrophotometrically and by 19F n.m.r. (nuclear magnetic resonance). The 19F n.m.r. line-width-pH and chemical-shift-pH profiles of difluoro-oxaloacetate in the presence of the aldimine form of the enzyme both show inflexion points in the pH5 and pH8 regions, which may arise from variations in the binding of difluoro-oxaloacetate as specific groups on the enzyme are successively protonated. Difluoro-oxaloacetate also interacts with apoenzyme to form a complex, the dissociation constant of which was determined by 19F n.m.r. The 19F n.m.r. line-width-pH and chemical-shift-pH profiles of difluoro-oxaloacetate in the presence of apoenzyme show a single inflexion point in the region of pH8. The absence, in this case, of an inflexion in the pH5 region indicates that the latter, present in the corresponding profiles for the aldimine form of the enzyme, results from ionization of an enzyme group associated with the pyridoxal phosphate cofactor.     

N.m.r. spectroscopy: A non-invasive tool for studying intracellular processes

The development of nuclear magnetic resonance (n.m.r.) spectroscopy as a non-invasive, non-destructive tool for the study of metabolic processes in cells and tissues is reviewed. Although n.m.r. measurements are subject to some limitations, e.g. low sensitivity and the need for special probes for in vivo work, the quality of n.m.r. instruments is steadily improving and wide bore spectrometers have been introduced. The article reviews in vivo n.m.r. studies of suspended and immobilized cells and also gives examples of the use of solid state n.m.r. Plant cell suspensions and tissues are good systems for study by n.m.r. as the P_i resonances from different compartments in the cell can be easily identified, and are important indicators of cellular metabolism. The literature on the use of magnetization transfer and on techniques for the study of enzyme catalysed reactions is also reviewed. Future applications of n.m.r. in examining biotechnological processes are discussed.     

H and 13C n.m.r. studies of serum from normal and Echinococcus multilocularis infected jirds

The major components of the 13C and high field region of the 1H nuclear magnetic resonance (n.m.r.) spectra of normal and Echinococcus multilocularis infected jirds were identified and compared. Substantial depletion of the glucose and fatty acid chains from lower density lipoproteins was detected in sera from infected animals. In addition, this proliferating metacestode markedly changed the appearance of the spectral region recently assigned to N-acetyl protons of carbohydrate side chains of N-acetylated glycoproteins.     

The fine structure of two DNA dodecamers containing the cAMP responsive element sequence and its inverse. Nuclear magnetic resonance and molecular simulation studies.

1H and 31P n.m.r. (nuclear magnetic resonance) spectroscopy have been used in conjunction with molecular simulation to determine the structure of two DNA dodecamers. The first of these, CATGACGTCATG, contains the octameric sequence CRE (cAMP responsive element), while the second is the reversed sequence, GTACTGCAGTAC. Structure determination was based on both NOESY (nuclear Overhauser spectroscopy) derived distances and COSY (correlated spectroscopy) dihedral angle data. Access to the 31P spectra also allowed the epsilon backbone angles to be determined. Considerable care was taken in deriving structural parameters from the n.m.r. data and an excellent level of agreement is obtained with the simulated conformations. Both dodecamers are found to belong to the B-DNA family; however, there is a striking difference between the CRE sequence and its inverse, the former conformation alone showing a strong structural heterogeneity.     

N.m.r. studies of metabolism in perfused organs

Several metabolites and intracellular pH in intact organs can be studied in a non-destructive manner by phorphorus nuclear magnetic resonance (31P n.m.r.). This possibility was demonstrated by us nearly five years ago. Since then we have developed the appropriate physiological techniques and improved the n.m.r. method for the study of animal hearts and kidneys. Here we described measurements aimed at clarifying three problesm. (1) Having measured the enzyme-catalysed fluxes between phosphocreatine and ATP by the method of saturation transfer n.m.r., we examine the relations between energy supply and heart rate in the isolated perfused rat heart. (2) We describe experiments to establish the validity of the perfusion model. For the first time, we report 31P n.m.r. measurements of an in vivo rat heart and compare the results with those obtained for the perfused rat heart. (3) Ischaemia and metabolism in rabbit kidneys is investigated to establish the relation between functional and metabolic recovery after a renal transplant operation.     

Type-specific polysaccharides of Cryptococcus neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.     

The Wellcome Foundation lecture, 1984. Nuclear magnetic resonance imaging in medicine: medical and biological applications and problems

From early biological work and the first T1 nuclear magnetic resonance (n.m.r.) animal image in 1974, whole-body patient images, by using a two-dimensional Fourier transform method were achieved in Aberdeen in 1980 with a 0.04 T vertical resistive magnet. Different pulse sequences produce images dependent by different amounts on proton density, T1 and T2, and for clinical work it is advantageous to use more than one pulse sequence to image pathology. The slow improvement of spatial resolution with increasing standing magnetic field strength is discussed and information on the T1 and T2 contrast dependence is reviewed: it suggests that the gains from high fields may be less than believed hitherto. Electrocardiogram gating can be used to produce moving images of the beating heart; blood flow can be imaged and surface radiofrequency coils are used for improved detail. N.m.r. imaging has considerable potential for studying response to therapy; mental states and dementia; tissue generation; discriminating body fat and body fluids. Other nuclei such as 23Na can be imaged and the potential to image fluorine-labelled pharmaceuticals could be very exciting; n.m.r. contrast agents are now being developed. Images formed from T1 values measured for each pixel are very useful for diagnosis, but the numerical values themselves are less valuable for distinctive pathological identification. With 15 companies manufacturing n.m.r. imagers and over 200 in use in hospitals, the technique is rapidly becoming established in diagnostic clinical practice and some typical uses are presented.     

The Wellcome Foundation lecture, 1981. Nuclear magnetic resonance imaging in medicine: physical principles

In recent years nuclear magnetic resonance (n.m.r.) has become a means of providing excellent images of the interior of the human body which are proving useful in medical practice. The development of n.m.r. imaging, much of which was pioneered in Britain, is outlined. Proton image resolution of human anatomy is comparable with X-ray computed tomography images, but without the hazard of ionizing radiation. There is improved soft tissue discrimination and pathological contrast through the basic imaging parameters of the proton density and the relaxation times T1 and T2, whose differences from one tissue to another are exploited by use of appropriate radiofrequency pulse sequences. Images may be obtained directly of transverse, coronal and sagittal sections of the head and body. Single slices or multiple slices may be imaged and imaging may be done in three dimensions. The lecture describes the more important imaging techniques and gives illustrative examples of images obtained. The efficient use of time in n.m.r. imaging is discussed, particularly mentioning the multiecho-multislice procedure and the development of real-time n.m.r. imaging. Magnetic field strengths in current use for proton n.m.r. imaging range from 0.02 to 2 T. At the lower end of the range resistive magnets are used, while for higher fields superconducting magnets are needed. A considerable improvement in image quality is obtained by use of special receiver coils.     

1H n.m.r. studies of insulin. Assignment of resonances and properties of tyrosine residues.

The assignment of the aromatic 1H n.m.r. resonances of the four tyrosine residues of bovine 2-zinc insulin is reported, based on double resonance techniques, use of Hahn spin echo pulse sequences and examination of specific derivatives nitrated at tyrosines A14 and A19 as well as des-(B26-B30)-insulin. Titration curves of the four tyrosine residues show that residues A14 and B16 have normal pK' values of 10.3-10.6 in solution, consistent with their accessibility to solvent in monomer and dimer in the crystal. Tyrosine residues A19 and B26 have pK' values of 11.4 and exhibit other features in their titration curves that are consistent with limited accessibility to solvent and a nonpolar environment. The meta protons of residues B16 and B26 both observe the titration of a nearby tyrosine residue, probably A19. Interpretation of the n.m.r. data obtained in solution is consistent with the crystallographic data for the monomer and dimer obtained on insulin crystals [Blundell, Dodson, Hodgkin & Mercola (1972) Adv. Protein Chem. 26, 279-402].     

Polypeptide fold in the two metal clusters of metallothionein-2 by nuclear magnetic resonance in solution

The solution conformation of rabbit liver Cd27+-metallothionein-2 was determined by nuclear magnetic resonance (n.m.r.) and distance geometry. The n.m.r. data are based on complete sequence-specific resonance assignments for the polypeptide chain. This letter describes the global arrangement of the polypeptide chain, which forms two distinct domains containing metal clusters of three and four Cd ions, respectively.     

Nuclear magnetic resonance study of the solution structure of alpha 1-purothionin. Sequential resonance assignment, secondary structure and low resolution tertiary structure

The solution structure of the 45-residue plant protein, alpha 1-purothionin, is investigated by nuclear magnetic resonance (n.m.r.) spectroscopy. Using a combination of two-dimensional n.m.r. techniques to demonstrate through-bond and through-space (less than 5 A) connectivities, the 1H n.m.r. spectrum of alpha 1-purothionin is assigned in a sequential manner. The secondary structure elements are then delineated on the basis of a qualitative interpretation of short-range nuclear Overhauser effects (NOE) involving the NH, C alpha H and C beta H protons. There are two helices extending from residues 10 to 19 and 23 to 28, two short beta-strands from residues 3 to 5 and 31 to 34 which form a mini anti-parallel beta-sheet, and five turns. In addition, a number of long-range NOE connectivities are assigned and a low resolution tertiary structure is proposed.     

Highly effective DNA in stimulating poly-(ADP-ribose) polymerase reaction.

The principle and the experimental realization of spin echo correlated spectroscopy (SECSY) are described and its use for studies of proteins is illustrated with ¹H n.m.r. spectra of the basic pancreatic trypsin inhibitor. This technique yields two-dimensional homonuclear correlated spectra which manifest connectivities between spin coupled nuclei and thus provide first level assignments of individual spin systems in biopolymers. Compared to previously described two-dimensional correlated n.m.r. techniques, which were applied exclusively to small molecules, SECSY uses a smaller data size both in the time domain and the frequency domain, which makes it particularly suitable for studies of macromolecules.     

Fluorescent probes in membrane studies.

A number of spectroscopic techniques are suitable for studying biological membranes. Of these, fluorescence has the sensitivity and time resolution for following membrane events associated with nerve excitation. In this paper, the nature of the information derived from measurements of the fluorescence properties of externally introduced chromophores in membranes is examined. In particular, the locations of various probes are described on the basis of nuclear magnetic resonance (n.m.r.) experiments in model situations. Then the motional characteristics of the probe molecules (rotation and diffusion) are discussed. Finally experiments designed to relate the detailed observations that can be made in lipid bilayers using n.m.r. and fuorescence measurements to those (more limited in nature) that can be made in membranes are described.