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J Feeney

Publications and source records attributed to J Feeney.

At least 55 records · Page 3Linked to original sources
PubMed1994

31P-NMR studies of NADPH, NADP+ and the complex of NADPH and methotrexate with Lactobacillus casei dihydrofolate reductase in the solid state.

31P-NMR spectra on solid samples of NADP+, NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross polarisation, magic angle spinning and high power proton decoupling. The isotropic chemical shifts, the principal components of the shielding tensors and the asymmetry parameters for the 31P nuclei in the 2'-phosphate and pyrophosphate groups have been measured. The isotropic shifts show similar trends to the chemical shifts measured in solution. The isotropic shifts and the shielding tensors for the dianionic and monoanionic states of the 2'-phosphate group have been determined and the presence of both ionisation states has been detected in a solid sample of the lyophilised complex of L. casei dihydrofolate reductase with NADPH and methotrexate. This contrasts with the behaviour in solution, where only the dianionic form is bound to the enzyme. The signals from the two pyrophosphates 31P nuclei in bound NADPH were resolved and identified. The asymmetry parameters in the different ionisation states and the orientations of the shielding tensors within the molecular framework are considered in the context of previous 31P studies on phosphate-containing compounds.

Lacticaseibacillus casei↗
PubMed1994

Solution structure of bound trimethoprim in its complex with Lactobacillus casei dihydrofolate reductase.

Two- and three-dimensional (2D and 3D) NMR techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (M(r) 18,300) in its 1:1 complex with the antibacterial drug trimethoprim. A sample of uniformly 15N-labeled protein was examined using 3D 15N/1H experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOEs between trimethoprim and protein protons and four intramolecular NOEs in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15N-and 13C-labeled trimethoprim molecules ([1,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound to unlabeled protein. The ligand-protein NOEs were used as distance constraints in conjunction with minimum energy and simulated annealing calculations (carried out with X-PLOR) to dock the trimethoprim ligand into dihydrofolate reductase, using as a starting structure the crystal coordinates from a related complex with a similar overall protein structure. The restrained minimum energy calculations and the simulated annealing calculations gave 83 calculated structures with distance violations of < 0.1 A. In all of these, the two aromatic rings of trimethoprim occupied essentially the same region of conformational space in the binding site (RMSD = 0.63 A). The protein residues nearest to the bound trimethoprim were found to be very similar in all of the structures and agreed well with corresponding contact residues observed in the X-ray crystal studies on trimethoprim complexes formed with Escherichia coli and chicken liver DHFRs.

Amino Acid Sequence↗
PubMed1994

3H-n.m.r. studies of multiple conformations and dynamic processes in complexes of folate and methotrexate with Lactobacillus casei dihydrofolate reductase.

[7,3',5'-3H3]- and [7,9-3H3]-folic acid and [7,3',5'-3H3]methotrexate (MTX) have been prepared and 3H-n.m.r. spectra obtained for their complexes with Lactobacillus casei dihydrofolate reductase (DHFR). The 3H results confirm the presence of three pH-dependent different conformational forms in the complex DHFR.NADP+.folate. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms, with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the MTX complex was shown to exist as a single conformational state with the benzoyl ring flipping rate being too low to give a single averaged signal for the 3',5'-nuclei over the temperature range 283-313 K.

Escherichia coli↗
PubMed1994

Solution structure of the active domain of tissue inhibitor of metalloproteinases-2. A new member of the OB fold protein family.

Homonuclear two-dimensional and three-dimensional 1H nuclear magnetic resonance spectroscopy has been used to obtain essentially complete sequence-specific assignments for 123 of the 127 amino acid residues present in the truncated form of tissue inhibitor of metalloproteinases-2 (delta TIMP-2), the active N-terminal domain of the protein. Analysis of the through-space nuclear Overhauser effect data obtained for delta TIMP-2 allowed determination of both the secondary structure of the domain and also a low-resolution tertiary structure defining the protein backbone topology. The protein contains a five-stranded antiparallel beta-sheet that is rolled over on itself to form a closed beta-barrel, and two short helices which pack close to one another on the same barrel face. A comparison of the delta TIMP-2 structure with other known protein folds reveals that the beta-barrel topology is homologous to that seen in proteins of the oligosaccharide/oligonucleotide binding (OB) fold family. The common structural features include the number of beta-strands and their arrangement, the beta-barrel shear number, an interstrand hydrogen bond network, the packing of the hydrophobic core, and a conserved beta-bulge. Superpositions of the beta-barrels from delta TIMP-2 and two previously known members of the OB protein fold family (staphylococcal nuclease and Escherichia coli heat-labile enterotoxin) confirmed the similarity in beta-barrel topology. The three-dimensional structure of delta TIMP-2 has allowed a more detailed interpretation than was previously possible of the functional significance of available protein sequence and site-directed mutagenesis data for the TIMP family. Furthermore, the structure has revealed conserved surface regions of potential functional importance.

Amino Acid Sequence↗
PubMed1994

Anti-HIV-1 activity of chemically modified heparins: correlation between binding to the V3 loop of gp120 and inhibition of cellular HIV-1 infection in vitro.

Chemically modified heparins were tested for their activities in (i) inhibiting HIV-1 replication in vitro and (ii) inhibiting the binding to recombinant HIV-1 gp120 of monoclonal antibodies specific for the V3 loop. The results reveal that N-desulfation reduces activity, although this is largely restored on N-acetylation. Selective O-desulfation also markedly reduces activity, whereas carboxyl reduction has little effect. Overall these results show that the anti-HIV-1 activity of heparin does not depend simply on negative density, and indicate instead that particular structures, notably O-sulfates, are involved. Our studies reveal that for chemically modified heparins and heparin-derived fragments there is a striking correlation between anti-HIV-1 activity in vitro and binding to the V3 loop of gp120 in solid phase ELISA. This strongly suggests that the heparin exerts its anti-HIV-1 activity by binding to the V3 loop of gp120.

Acetylation↗
PubMed1994

Solution structure of a trefoil-motif-containing cell growth factor, porcine spasmolytic protein.

The porcine spasmolytic protein (pSP) is a 106-residue cell growth factor that typifies a family of eukaryotic proteins that contain at least one copy of an approximately 40-amino acid protein domain known as the trefoil motif. In fact, pSP contains two highly homologous trefoil domains. We have determined the complete three-dimensional solution structure of pSP by using a combination of two- and three-dimensional 1H NMR spectroscopy and distance geometry calculations. pSP is a relatively elongated molecule, consisting of two compact globular domains joined via a small interface. The protein's two trefoil domains adopt the same tertiary structure and contain a core C-terminal two-stranded antiparallel beta-sheet, preceded by a 6-residue helix that packs against the N-terminal beta-strand. The remainder of the protein backbone is taken up by two short loops that lie on either side of the beta-hairpin and are linked by an extended region that wraps around the C-terminal beta-strand. The topology of the protein backbone observed for the trefoil domains in pSP represents an unusual polypeptide fold. A striking feature of both trefoil domains is a surface patch formed from five conserved residues that have no obvious structural role. The two patches are located at the far ends of the protein molecule, and we propose that these residues form at least part of the receptor binding site, or sites, on pSP.

Amino Acid Sequence↗
PubMed1994

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase.

A mutant of Lactobacillus casei dihydrofolate reductase has been constructed in which Thr63, a residue which interacts with the 2'-phosphate group of the bound coenzyme, is replaced by alanine. This substitution does not affect kcat, but produces an 800-fold increase in the Km for NADPH, which reflects dissociation of NADPH from the enzyme-NADPH-tetrahydrofolate complex, and a 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociation constant for the enzyme-NADPH complex. The difference in magnitude of these effects indicates a small effect of the substitution on the negative cooperativity between NADPH and tetrahydrofolate. Stopped-flow studies of the kinetics of NADPH binding show that the weaker binding arises predominantly from a decrease in the association rate constant. NMR spectroscopy was used to compare the structures of the mutant and wild-type enzymes in solution, in their complexes with methotrexate and with methotrexate and NADPH. This showed that only minimal structural changes result from the mutation; a total of 47 residues were monitored from their resolved 1H resonances, and of these nine in the binary complex and six in the ternary differed in chemical shift between mutant and wild-type enzyme. These affected residues are confined to the immediate vicinity of residue 63. There is a substantial difference in the 31P chemical shift of the 2'-phosphate of the bound coenzyme, reflecting the loss of the interaction with the side chain of Thr63. The only changes in nuclear Overhauser effects (NOEs) observed were decreases in the intensity of NOEs between protons of the adenine ring of the bound coenzyme and the nearby residues Leu62 and Ile102, showing that the substitution of Thr63 does cause a change in the position or orientation of the adenine ring in its binding site.

Alanine↗
PubMed1993

Conformational differences between complexes of elongation factor Tu studied 19F-NMR spectroscopy.

An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine. The 19F-NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF-Ts) and the ternary complexes EF-Tu.GDP.aurodox and EF-Tu.GDP.EF-Ts were measured. EF-Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances. EF-Tu.GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large downfield shifts can be explained by second-order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF-Tu.GDP and with the steric restraints being released in all of the other complexes. The X-ray diffraction structure of EF-Tu.GDP shows that Tyr87 in the N-terminal domain (domain I) and Tyr309 in the C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural changes between EF-Tu.GDP and EF-Tu.GTP by other workers. If these tyrosine residues correspond to the two downfield resonances of the spectra of EF-Tu.GDP, the results from the 19F-NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF-Tu.GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction. For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains. The 19F-NMR spectrum of the trypsin-cleaved product of EF-Tu.GDP, from which the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.

Aurodox↗
PubMed1993

13C NMR determination of the tautomeric and ionization states of folate in its complexes with Lactobacillus casei dihydrofolate reductase.

13C NMR studies provide a convenient way of obtaining detailed information about tautomeric and ionization states in protein-ligand complexes provided that suitably 13C-labeled molecules are available. In the present study, [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid were synthesized and the 13C NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analyzed as a function of pH. From these data it was possible to determine the tautomeric and ionization states of the bound folate and to obtain further evidence about the orientation of the pteridine ring in the complexes. In the 13C spectra of the ternary complexes of the 13C-labeled folic acids with DHFR and NADP+, each labeled carbon gave rise to multiple signals, confirming our previous findings that there are three interconverting conformational forms of bound folate (forms I, IIa, and IIb) in the ternary complex (Birdsall et al., 1989b). The 13C spectra of the binary complexes of folate and DHFR also provide direct evidence for the presence of forms IIa and IIb and indirect evidence of some form I at low pH values ( < 5.0). 2D 1H-13C HMQC-NOESY experiments on ternary complexes formed using the [2,4a,7,9-13C]folic acid were used to obtain intermolecular NOEs between the folate H7 proton and protons on the protein, and these provided further characterization of the orientations of the pteridine ring in the different bound forms of folate (form IIb with its pteridine ring in the catalytically active conformation and forms I and IIa with their pteridine rings turned over by 180 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1993

Stereospecific assignments of the leucine methyl resonances in the 1H NMR spectrum of Lactobacillus casei dihydrofolate reductase.

A general method is described for the stereospecific assignment of methyl resonances in protein NMR spectra based on selective deuteration procedures. A selectively deuterated dihydrofolate reductase from L. casei was prepared by incorporating stereoselectively deuterated L-leucine, (2S,4R)[5,5,5-2H3]leucine. By comparing the COSY spectra of the dihydrofolate reductase-methotrexate complexes formed using deuterated and non-deuterated enzyme the stereospecific assignments for resonances of all 13 leucine residues were obtained by noting the absence of cross-peaks in spectra from the deuterated proteins.

Bacterial Proteins↗
PubMed1992

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.

Escherichia coli↗
PubMed1992

19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli.

A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.

Bacterial Proteins↗
PubMed1992

Characterisation by mass spectrometry and 1H-NMR of novel hexasaccharides among the acidic O-linked carbohydrate chains of bovine submaxillary mucin.

The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characterised in the neutral chains of bovine submaxillary-gland mucin.

Animals↗
PubMed1992

Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution.

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.

Binding Sites↗
PubMed1992

31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments.

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.

Lacticaseibacillus casei↗
PubMed1992

Neutral oligosaccharides of bovine submaxillary mucin. A combined mass spectrometry and 1H-NMR study.

Twenty-two neutral O-linked oligosaccharides ranging from monosaccharides to octasaccharides were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3-linked to N-acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(beta 1-6) [Gal(beta 1-3)]GalNAcol or GlcNAc(beta 1-6)[GlcNAc(beta 1-3)]GalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAc alpha 1-6GalNAcol (GalNAc, N-acetylgalactosamine and the hexasaccharide GlcNAc(beta 1-6) [GalNAc(alpha 1-3)( Fuc (alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-3)]GalNAcol (Fuc, L-fucose). Oligosaccharides of the blood-group-A type have not been detected previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric-mucosal glycoproteins has been established by classical immunochemical studies.

ABO Blood-Group System↗