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I M Armitage

Publications and source records attributed to I M Armitage.

At least 37 records · Page 2Linked to original sources
PubMed1993

Copper- and silver-substituted yeast metallothioneins: sequential 1H NMR assignments reflecting conformational heterogeneity at the C terminus.

Complete 1H NMR sequential assignments have been made for copper(I)- and silver (I)-substituted metallothionein (MT) from Saccharomyces cerevisiae using standard 2D 1H NMR methods. The fingerprint region of the COSY spectrum of both metalloproteins shows a doubling of a few backbone proton resonances from residue K41 onward in the C terminus. This doubling of resonances is absent in the spectrum of the truncated mutant protein that lacks the five C-terminal residues which includes two cysteines. Concurrently, it has been established from a comparison of the heteronuclear 1H-109 Ag multiple-quantum coherence transfer (HMQC) spectrum on the silver-substituted mutant and the wild-type protein that metal ligation is similar in both molecules. Thus, the 2 C-terminal Cys are not essential for metal cluster formation in the wild-type yeast MT and only 10 of the 12 Cys present in this protein appear to be involved in ligating the 7 mol of bound metal ions. A qualitative analysis of the coupling constant, hydrogen exchange, and NOE data indicates the presence of many type I beta-turns and the lack of any other regular secondary structural elements. A comparison of chemical shifts and NOE data for native copper- and silver-substituted yeast MT indicates a high degree of conservation of structural elements in both proteins. Therefore, it seems reasonable to conclude that the metal to Cys connectivities which are obtained directly from the HMQC data on silver-substituted metallothionein are conserved in the native copper protein. Interestingly, a mixture of both 2 and 3 coordination was found for the bound Ag(I) ions in a single Ag7Cys10 cluster. This mixed coordination number and a single cluster arrangement is most probably also shared with the Cu(I) ion coordination in the native protein.

Amino Acid Sequence↗
PubMed1993

Biologically active Arg-Gly-Asp oligopeptides assume a type II beta-turn in solution.

The sequence Arg-Gly-Asp (RGD) has been found to be the consensus sequence of matrix proteins for binding cell surface receptors (integrins). Studies with synthetic peptides containing the RGD sequence show that the biological activity of these oligopeptides is removed upon a conservative substitution of Glu for Asp in the RGD sequence. Two-dimensional 1H NMR methods were used to investigate the secondary structures in aqueous solution for two such oligopeptides of differing biological activity. The sequence Tyr-Gly-Arg-Gly-Asp-Ser-Pro, which binds to selected integrins, is found to assume a type II beta-turn at both pH 4 and 7. In contrast, the sequence Tyr-Gly-Arg-Gly-Glu-Ser-Pro, which does not interfere with integrin-mediated cell attachment, is found to assume a type I or III beta-turn at both pH 4 and 7. This comparison confirms not only that oligopeptides can assume a secondary structure in aqueous solution, but also that these structures may be important to biological functions.

Amino Acid Sequence↗
PubMed1992

Solution structure of cyclosporin A and a nonimmunosuppressive analog bound to fully deuterated cyclophilin.

A simple strategy involving 1H nuclear magnetic resonance (NMR) spectroscopy and complete protein deuteration was used to determine the structures of two receptor-bound drugs. A potent immunosuppressive, cyclosporin A (CsA) binds tightly to the ubiquitous and highly conserved 17.7-kDa immunophilin, cyclophilin (CyP). Fully deuterated CyP was produced by overexpressing the human CyP gene in Escherichia coli grown on deuterated algal hydrolysate in 98% D2O. As only the CsA molecule is protonated in the CsA-CyP complex, we were able to make a complete sequential assignment of the bound drug using standard two-dimensional proton NMR experiments. The structure determination was accomplished using dynamical simulated annealing calculations with a total of 124 NMR-derived distance and torsion angle restraints. Aside from binding CsA, CyP also acts as a peptidyl-prolyl cis-trans isomerase. Thus, much importance had been ascribed to the cis peptide bond present in the structures reported for free CsA in organic solvents and in crystal studies. Interestingly, CyP-bound CsA exists in an all-trans conformation with no detectable elements of regular secondary structure and no intramolecular hydrogen bonds. A nonactive CsA analog, MeAla6-CsA, was studied using the same CyP deuteration strategy. In addition to structural elucidation of the two bound drugs, we were able to differentiate between the bound and surface-exposed residues of the drugs and also validate our previous hypothesis that the single CyP tryptophan is located in the CsA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗
PubMed1991

Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.

Animals↗
PubMed1991

Conformation of MeAla6-cyclosporin A by NMR. Relationship of sidechain orientation of the MeBmt-1, MeLeu-9, and MeLeu-10 residues to immunosuppressive activity.

MeAla6-cyclosporin A (MeAla6-CsA) is a unique CsA analog that shows weak immunosuppressive activity and yet binds strongly to the proposed cytosolic protein receptor, cyclophilin (CyP). Preliminary 1H NMR data showed significant chemical shift differences between spectra of MeAla6-CsA and CsA, suggesting different preferred conformations. A more detailed study, however, revealed that the backbone conformations of the two molecules are essentially identical, and that the differences can be accounted for, principally, by the sidechain motions of the MeBmt-1, MeLeu-9, and -10 residues. ROE and coupling constant data show that in MeAla6-CsA, the preferred chi 1 rotamers for MeLeu-9 and -10 are + 180 degrees (T), whereas in CsA there is a more even distribution of rotamer populations for MeLeu-10, and a preferred -60 degrees (G-) chi 1 rotamer for MeLeu-9. Similar data argue that the sidechain of MeBmt-1 is more restricted in its motion in MeAla-CsA than in CsA. Temperature studies suggest that these preferred rotamers for MeAla6-CsA may increase the stability of the hydrogen bond between NH(7) and CO(11), but prevent particular residues, especially the essential MeBmt-1 sidechain, from adopting orientations required to elicit immunosuppressive activity. The significant changes observed in the preferred orientations for the sidechains of the MeBmt-1, MeLeu-9, and MeLeu-10 residues in MeAla6-CsA argue that the particular orientations which they assume in CsA are not essential for cyclophilin binding.

Amino Acid Sequence↗
PubMed1990

A binding site peptide fragment of the nicotinic acetylcholine receptor. Sequence-specific assignment of 1H-NMR resonances in the dodecamer alpha 185-196.

The total sequence-specific 1H assignment for the alpha 185-peptide was accomplished by analysis of COSY spectra along with spin-decoupling and confirmatory NOE difference experiments. Some ambiguities in the assignments were successfully addressed utilizing additional peptides with selective amino acid substitutions. The chemical shifts of several of the C alpha H resonances, along with evidence for a slowly exchanging amide at Thr-191 suggest that the alpha 185-peptide may contain a certain amount of non-random coil structure. The role of any such ordered structure in the mechanism of binding to alpha-bungarotoxin remains to be determined. The assignment of the peptide 1H resonances will facilitate the analysis and identification of chemical shift perturbations observed upon formation of the complex between alpha-bungarotoxin and the alpha 185-peptide [7].

Amino Acid Sequence↗
PubMed1990

Structural elements pertinent to the interaction of cyclosporin A with its specific receptor protein, cyclophilin.

Cyclophilin (163 amino acids; 17,737 daltons) is a ubiquitous cytosolic protein that specifically binds the potent immunosuppressive drug cyclosporin A (CsA). To characterize the structural details of this interaction, extensive use has been made of two-dimensional (2D) NMR methods. For studies on CsA, these methods are being used to assign the conformational space accessible to CsA by analysis of the spectra from the multiple CsA conformers present in slow exchange in mixed solvent systems. These same 2D NMR methods also have been used for extensive studies of the major bovine thymus cyclophilin (CyP) isoform and its complex with stoichiometric amounts of CsA. In the former case, these studies have revealed 81% of the 156 expected HN-H alpha crosspeaks. The complete spin-coupled spin systems for one-third of these amide resonances have been assigned according to amino acid type. After exhaustive D2O exchange, there remain 44 amide protons which exhibit 2D NMR features indicative of a hydrophobic domain with beta-sheet secondary structure. The CsA-complexed form of CyP exhibits a discrete structure and set of resonances in slow exchange with the drug-free CyP. The amino acids that have been specifically identified to be affected by the interaction are limited in number and include three Phe residues, the unique Trp at position 120, and two Ala residues.

Amino Acid Sequence↗
PubMed1990

1H NMR studies on bovine cyclophilin: preliminary structural characterization of its complex with cyclosporin A.

Cyclophilin (163 residues, Mr 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/alpha CH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/alpha CH, and alpha CH/alpha CH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a beta-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their beta CH's, main-chain alpha CH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled X11, which shows a close spatial relationship to the perturbed Phe12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Quantitative studies of hydrodynamic effects and cross-relaxation in protein solutions and tissues with proton and deuteron longitudinal relaxation times.

Longitudinal relaxation times T1 of water protons were measured in 5% protein solutions at different static magnetic fields (0.47, 2, and 7 T), for proteins with molecular weight ranging between 1.4 and 480 kDa and in solvents of varying degrees of deuteration. T1 values were also obtained for rat liver soaked with Krebs-Ringer solutions of varying degrees of deuteration at the above fields. For the samples containing D2O, T1 for deuterium was also measured at fields 2 and 7 T. The deuterium measurements were used to estimate water rotational correlation times which were in turn used to estimate the contribution of so-called "hydrodynamic effects" of macromolecules to proton relaxation. The proton relaxation rates at full deuteration were compared with those in protonated solvent (water) to obtain a second, direct measurement of this effect. Both measurements provide quantitation of the hydrodynamic effects, free from the contributions of other effects that are transparent to deuteration, and results from both measurements agree with each other reasonably well. The cross-relaxation rate between solute and solvent protons, and the contribution of paramagnetic impurities in the samples were also obtained from the proton T1 studies. The experimental results show that the hydrodynamic effects (intramolecular and intermolecular water-water interactions) are about the same magnitude in all the proteins studied as well as in rat liver. However, the cross-relaxation rate generally increases with increasing protein molecular weight. Measurements in soaked rat liver indicate that the cross-relaxation rate per unit mass of solute is much higher in tissues than in simple solutions of proteins of similar mean molecular weight. The results challenge the prevailing concept that the relaxation properties of biological tissues may be treated as a simple superposition of the properties of their constituents.

Animals↗
PubMed1989

Proton NMR studies of a metallothionein from Neurospora crassa: sequence-specific assignments by NOE measurements in the rotating frame.

Sequential 1H NMR assignments of a metallothionein from Neurospora crassa have been accomplished by the combined use of COSY, 2QF-COSY, HOHAHA, and rotating-frame NOE experiments. All potentially observable resonances were assigned except for the epsilon-NH3 group of the C-terminal lysine. 1H NOEs, when observed in the laboratory frame and at 500-MHz spectrometer frequency, were negligible in this protein due to the inherent rotational correlation time of the molecule. This difficulty was circumvented by measuring transverse NOEs in the rotating frame under spin-locking conditions. The observed pattern of NOEs reveals a marked absence of "regular" secondary structures in the protein. Thus, the stability of this metallothionein's tertiary structure must arise primarily from its metal ligation. This appears to be a general feature of MTs since a general lack of extensive secondary structural elements was also observed in other metallothioneins.

Amino Acid Sequence↗
PubMed1989

NMR spectroscopy of 113Cd(II)-substituted gene 32 protein.

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 113Cd resonance, delta 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a delta and a line width the same as those observed for the wild-type protein. Thus, the S-ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligand protons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions, respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cadmium↗
PubMed1989

An analysis of magnetic cross-relaxation between water and methylene protons in a model system.

Experiments have been performed to demonstrate and quantify cross-relaxation between water and methylene resonances in a simple model system. Inversion recovery experiments on aqueous solutions of polyethylene glycol produce a transient nuclear Overhauser effect that alters the recovery of the methylene resonance. The relevant equations and parameters that describe this effect are identified and their values are explicitly quantified by analyzing the experimental results. It is shown that cross-relaxation is measurable and significant in this system even though there are no strong hydrophilic interactions available to immobilize water. In studies of dog bile, the water and lipid resonances behave in a qualitatively similar fashion, lending support to the contention that cross-relaxation cannot be neglected in solutions of biological macromolecules.

Animals↗
PubMed1989

Relative contributions of chemical exchange and other relaxation mechanisms in protein solutions and tissues.

Transverse relaxation times T2 of water protons were measured in 5% protein solutions and soaked rat liver in different static magnetic fields (0.15 to 11 T). Protein molecular weight varied between 1.4 and 480 kDa in solutions of varying degrees of deuteration. The data obtained are analyzed in terms of a model system consisting of three phases of different relaxation characteristics: protein protons, hydration layer water protons, and bulk water protons. The contributions to relaxation due to hydrodynamic effects on water protons, cross relaxation between the hydration layer water protons and the protein protons, and chemical exchange between the hydration layer water protons and the bulk water protons are separately estimated. The experimental results indicate that the "hydrodynamic interactions" are about the same magnitude in rat liver and all the proteins studied, but the contribution of the cross relaxation differs by several orders in different protein systems. Fast chemical exchange between the hydration layer water and the bulk water causes considerable shortening of T2 at high magnetic fields for all the protein solutions and rat tissue studied. Selected samples were studied at different temperatures (213-318 K) and with different intervals in the CPMG sequence. The rates of chemical exchange and fractional populations of different phases are determined, and the results obtained provide support for the model in which fast exchange among the different water phases is an important feature of the overall relaxation behavior.

Animals↗
PubMed1988

Magnetic resonance, pathology and physiology of the BA1112 rhabdomyosarcoma in vivo.

We studied histology, findings on H-1 magnetic resonance (MR) imaging, and correlations of P-31 MR spectroscopy with microelectrode pH and pO2 measurements in the BA1112 rhabdomyosarcoma in WAG/Rij/Y rats. Intratumoral hemorrhage was a prominent feature on MR images and pathologic specimens. Eosinophilic necrosis could be seen microscopically but was not discernible on images. The peaks seen on P-31 MR spectra were similar to those reported in other tumors. The intratumoral pH was neutral despite low pO2 values and P-31 MR evidence for impaired metabolic status.

Animals↗
PubMed1988

Nuclear magnetic resonance spectroscopy of skin: predictive correlates for clinical application.

The first application of phosphorous 31 (31P) and proton (1H) nuclear magnetic resonance (NMR) spectroscopy to the analysis of the metabolic profiles of skin flaps in a rat model and of human skin grafts is presented. Resonances of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi) were identified in 31P nuclear magnetic resonance spectra. Resonances of phosphocreatine, creatine (Cr), and lactate (Lac) were identified in 1H nuclear magnetic resonance spectra. The most significant finding was the substantial presence of phosphocreatine as the major high-energy phosphometabolite in mammalian skin, a finding which heretofore has not been widely recognized. An energy shuttle between phosphocreatine and ATP is operative in skin to buffer the fall in ATP during ischemic (anaerobic) insult. Inability to replenish exhausted phosphocreatine reserves predictively correlates with eventual flap necrosis. We have defined and analyzed temporal fluxes in the phosphocreatine-creatine and phosphocreatine plus creatine-lactate ratios by proton nuclear magnetic resonance. Both are sensitive, accurate, and unambiguous early prognostic indices of eventual flap outcome. These findings support the concept that the fate of a flap may be established as early as 3 hours after elevation and have laid the groundwork for development and application of noninvasive in vivo nuclear magnetic resonance spectroscopy to the study of skin flaps in animals and humans.

Adenosine Triphosphate↗
PubMed1987

NMR analysis of the structure and metal sequestering properties of metallothioneins.

Multinuclear 1 and 2 dimensional magnetic resonance methods have been used to investigate the structures and metal binding properties of metallothioneins (MTs) isolated from several different sources. 113Cd NMR studies have unambiguously shown that the 7 g-atoms of Cd2+ bound per mole of the mammalian MT are located in two separate metal clusters, one containing 4 metal ions and the other, 3 metal ions. In the invertebrate (Scylla serrata) MT, similar studies have revealed that the 6 g-atoms of bound Cd2+ are distributed in two distinct 3-metal clusters while in Neurospora MT, the 3 g-atoms of bound Cd2+ are arranged in a pseudo 3-metal cluster. With the exception of one of the Cd2+ sites in this latter cluster, all the Cd2+ ions are tetrahedrally coordinated to four cysteine thiolate ligands with single cysteinyl sulfurs bridging adjacent metals. These conclusions are based on the 113Cd chemical shift data and a detailed analysis of the observed 113Cd-113Cd scalar couplings by both homonuclear decoupling and 2D techniques. In addition, the 113Cd NMR studies have revealed significant differences in the affinity of different metal ions for the two mammalian metal clusters. For the 3-metal cluster, the affinity is found to decrease in the order Cu+ greater than Cd2+ greater than Zn2+ with Cd2+ greater than Zn2+ for the 4 metal cluster and Cd2+ (4-metal cluster) greater than Cd2+ (3-metal cluster). The 113Cd NMR data are currently being integrated with 500 MHz 2D 1H and 1H-113Cd chemical shift correlated multiple quantum data sets to more completely define the structural arrangement of the metal clusters in the tertiary structure of these proteins.

Amino Acid Sequence↗
PubMed1987

Augmentation of experimental skin flap survival via postoperative phosphocreatine replacement.

Despite improvements in our understanding of cutaneous vascular territories, clinical skin flap necrosis resulting from ischemic compromise is still a reality. Therapy with vasodilators has been generally unsuccessful, but replacement of high-energy phosphometabolites through the use of ATP-MgCl2 and fructose 1,6-diphosphate has been effective. Recently we reported that phosphocreatine is the major high-energy phosphometabolite in mammalian skin and that ATP levels and cellular well-being in skin flaps are dependent on adequate supply of this phosphometabolite. We report herein the successful augmentation of survival of ischemically compromised skin flaps through postoperative phosphocreatine infusion. This metabolite effectively circumvents the nonfunctioning mitochondrial creatine-phosphocreatine energy shuttle without disturbing the delicate [ATP]/[ADP] balance in the cytosol. In addition, phosphocreatine may favorably redistribute blood flow from muscle to the ischemically compromised skin.

Animals↗