Biomedical subjects

G C Roberts

Publications and source records attributed to G C Roberts.

At least 127 records · Page 7Linked to original sources

PubMed1986

Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim.

We have measured the 13C chemical shifts for trimethoprim molecules selectively enriched with 13C at the 2-, 4-, 5-, 6-, and 7-positions and the p-OCH3 position in their complexes with Lactobacillus casei dihydrofolate reductase in the presence and absence of coenzyme analogues. The C2 carbon shifts indicate that the pyrimidine ring is protonated at N1 in all the complexes of trimethoprim with the enzyme and coenzymes and in each case the pyrimidine ring is binding in a similar way to that of the corresponding part of methotrexate in the enzyme-methotrexate complex. The C6 carbon of trimethoprim shows a large upfield shift in all complexes (3.51 to 4.70 ppm) but no shift in the complex of 2,4-diaminopyrimidine with the enzyme: these shifts probably arise from steric interactions between the C1' and C2' carbons and the H6 proton, which approach van der Waals contact in the folded conformation adopted by trimethoprim when bound to the enzyme. The large shift observed for C6 in all complexes indicates that the basic folded conformation is present in all of them. A comparison of the 13C shifts in the enzyme-trimethoprim-NADPH complex with those in the enzyme-trimethoprim binary complex shows substantial changes even for carbons such as C6 and p-OCH3 (0.46 and -0.36 ppm, respectively), which are remote from the coenzyme: these are caused by ligand-induced conformational changes that may involve displacement of the helix containing residues 42-49.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1986

13C-NMR and transient kinetic studies on lactate dehydrogenase [Cys(13CN)165]. Direct measurement of a rate-limiting rearrangement in protein structure.

Chemical modification of cysteine-165 in pig heart lactate dehydrogenase to produce lactate dehydrogenase [Cys(13CN)165] introduces an covalently bound, enriched 13C probe at a position adjacent to the active cen. The signal from the thiocyanate probe is clearly visible at 47 ppm relative to dioxane. On formation of binary complexes with NAD+ and NADH, no signal change is detected. Formation of the ternary complexes E-NADH-oxamate and E-NAD+-oxalate results in an upfield shift of the signal of 1.2 ppm. These results interpreted as demonstrating that binding of the substrate analogue induces a conformational change a position adjacent to the active centre. Exchange experiments in which the enzyme is poised in dynamic equilibrium between binary and ternary complexes show that the rate at which the probe senses a change environment is the same as the kinetically observed unimolecular event which limits the enzyme-catalyst reduction of pyruvate. The two processes show the same dependence on temperature, solvent composition and pH. These results indicate that the rate-limiting isomerisation corresponds to a rearrangement of the protein in the region of cysteine-165.

Amino Acid Sequence↗

PubMed1986

Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes.

Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme-methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49).

Amino Acid Sequence↗

PubMed1986

Identification of the 1H resonances of valine and leucine residues in dihydrofolate reductase by using a combination of selective deuteration and two-dimensional correlation spectroscopy.

Lactobacillus casei dihydrofolate reductase (Mr 18 500) contains 16 valine and 14 leucine residues. By comparing the 2D COSY NMR spectra of normal and [gamma-2H6]valine enzyme we have been able to identify all 60 methyl resonances from these residues, and to connect the pairs arising from the same residue. This pairing of the methyl resonances was aided by the examination of the 2D RELAY spectrum which also allowed the C alpha H resonances (and hence the complete spin systems) of 14 of the valine residues to be identified. The combination of selective deuteration with 2D NMR techniques is shown to be a powerful general method for resolving 1H resonances in the complex spectra of proteins and for assigning them to amino-acid type.

Deuterium↗

PubMed1985

A 1H-n.m.r. study of casein micelles.

The 1H-n.m.r. spectrum of casein micelles consists of a small number of moderately sharp (linewidth approx. 60 Hz) resonances superimposed on the envelope of very broad lines expected for particles of this size. These sharp lines resemble, in chemical shift and relative intensity, the spectrum of the isolated 'macropeptide' released from the micelles by treatment with chymosin. The sharp lines in the casein micelle spectrum are further sharpened by addition of chymosin and broadened markedly by addition of ethanol. These observations are consistent with the proposal that the 'macropeptide' (the C-terminal 64 residues of K-casein) forms flexible 'hairs' on the surface of the micelles.

Animals↗

PubMed1985

1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase.

The binding of trimethoprim and [1,3,2-amino-15N3]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by 15N and 1H NMR spectroscopy. 15N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The 15N chemical shifts and 1H-15N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme. The N1-proton resonance in the complex has been assigned using the 15N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160 +/- 10 S-1 at 313 K, with an activation energy of 75 (+/-9) kJ X mole-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.

Dimethyl Sulfoxide↗

PubMed1984

The combined use of selective deuteration and double resonance experiments in assigning the 1H resonances of valine and tyrosine residues of dihydrofolate reductase.

Selective deuteration is a general solution to the resolution problem which limits the application of double resonance experiments to the assignment of the 1H NMR spectra of proteins. Spin-decoupling and NOE experiments have been carried out on Lactobacillus casei dihydrofolate reductase and on selectively deuterated derivatives of the enzyme containing either [gamma-2H6]Val or [alpha, delta 2, epsilon 1-2H3]His, [alpha, delta 1, delta 2, epsilon 1, epsilon 2, zeta-2H6]Phe, [alpha, delta 1, epsilon 3, zeta 2, zeta 3, eta 2-2H6]Trp and [alpha, epsilon 1, epsilon 2-2H3]Tyr. When combined with ring-current shift calculations based on the crystal structure of the enzyme, these experiments allow us to assign 1H resonances of Val 61, Val 115, Tyr 46 and Tyr 68.

Deuterium↗

PubMed1984

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex.

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.

Carbon Isotopes↗

PubMed1984

19F-n.m.r. studies of 3',5'-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes.

19F-n.m.r. spectroscopy was used to study the binding of 3',5'-difluoromethotrexate to dihydrofolate reductase (tetrahydrofolate dehydrogenase) from Lactobacillus casei. The benzoyl ring of the bound difluoromethotrexate was found to 'flip' about its symmetry axis, and the rate (7.3 X 10(3) s-1 at 298 K) and activation parameters for this process were determined by lineshape analysis of the 19F-n.m.r. spectrum at a series of temperatures in the range 273-308 K. The contributions to the barrier for this process are discussed. Addition of NADP+ or NADPH to form the enzyme-difluoromethotrexate-coenzyme ternary complex led to an increase in the rate of benzoyl ring flipping by a factor of 2.6-2.8-fold, and to substantial changes in the 19F-n.m.r. chemical shifts. The possible nature of the coenzyme-induced conformational changes responsible for these effects is discussed.

Folic Acid Antagonists↗

PubMed1984

Domain structure and 1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.

Chemical Phenomena↗

PubMed1984

A 1H NMR study of the interactions and conformations of rationally designed brodimoprim analogues in complexes with Lactobacillus casei dihydrofolate reductase.

A consideration of the detailed structural information available from X-ray crystallographic and NMR studies on complexes of dihydrofolate reductase with inhibitors has led to the design of trimethoprim analogues with improved binding properties. Computer graphic techniques have been used to predict which substituent groups were required at the 3'-O position of brodimoprim (2,4-diamino-5-(3,5-dimethoxy-4-bromobenzyl)pyrimidine) to make additional interactions with the enzyme. NMR spectroscopy provided a convenient method of assessing if the analogues were binding in the predicted manner. On the basis of this approach, the C4,C6-dicarboxylic acid analogue IX was designed to interact with Arg-57 and His-28 in the enzyme, and this analogue was found to bind 3 orders of magnitude more tightly than the parent brodimoprim.

Binding Sites↗

PubMed1984

A 1H n.m.r. study of the role of the glutamate moiety in the binding of methotrexate to Lactobacillus casei dihydrofolate reductase.

The binding of a series of amide derivatives of methotrexate to Lactobacillus casei dihydrofolate reductase has been studied by inhibition constant measurements and by 1H n.m.r. spectroscopy. Amide modification of the alpha-carboxylate of methotrexate was found to prevent interaction of the gamma-carboxylate with the imidazole of His 28. Estimates of the contributions to the binding energy from the alpha-carboxylate-Arg 57 and gamma-carboxylate-His 28 interactions have been made from a combination of inhibition and n.m.r. data.

Folic Acid Antagonists↗

PubMed1984

[Identification of an active site in luteotropin (LH) and chorionic gonadotropin (CG) based on studies of their conformation in solution by proton nuclear magnetic resonance (NMR) and on the receptor-binding properties of hormone fragments].

Using porcine LH, the presence of a site of interaction with the specific testicular receptor of these hormones was demonstrated in the carboxyterminal part of the alpha subunit. This conclusion is based on results of studies of chemical mutilations and conformation in solution of these hormones and of their subunits by NMR. A NMR study of human CG showed that this was also applicable to this hormone. The synthetic peptide Tyr-Tyr-His-Lys-Ser, which corresponds to the carboxyterminal part of the alpha subunit does not demonstrate high affinity for binding to the receptor. These findings indicate that the formation of one of the active sites of these gonadotropins depends necessarily on their quaternary structure. The peptide corresponding to the site of uptake must be bound within the hormonal alpha beta complex to enable effective interaction with the receptor.

Animals↗

PubMed1983

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.

Guanidine↗

PubMed1983

Trimethoprim binding to bacterial and mammalian dihydrofolate reductase: a comparison by proton and carbon-13 nuclear magnetic resonance.

The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells has been studied by measuring the changes in chemical shift of nuclei of the ligand that accompanying binding. The 6- and 2',6'-proton chemical shifts of bound trimethoprim have been determined by transfer of saturation experiments, and the 2-carbon chemical shift has been determined by using [2-13C]trimethoprim. The changes in proton chemical shift are substantially smaller than those accompanying binding to bacterial dihydrofolate reductase [Cayley, P. J., Albrand, J. P., Feeney, J., Robert, G. C. K., Piper, E. A., & Burgen, A. S. V. (1979) Biochemistry 18, 3886]. It is shown that this difference arises largely from the fact that trimethoprim adopts different conformations when bound to mammalian and to bacterial dihydrofolate reductase. The proton chemical shifts are interpreted in terms of ring-current contributions from the two aromatic rings of trimethoprim itself and the nearby aromatic amino acid residues of the enzyme. The latter have been located by using the refined crystallographic coordinates of the Lactobacillus casei and Escherichia coli reductases in their complexes with methotrexate [Bolin, J. T., Filman, D. J., Matthews, D. A. & Kraut, J. (1982) J. Biol. Chem. 257, 13650], under the assumption that, as indicated by the 13C chemical shifts, the diaminopyrimidine ring of trimethoprim binds in the same way as does the corresponding part of methotrexate. With use of these assumptions, the conformation of trimethoprim bound to the dihydrofolate reductases from L. casei, E. coli, and L1210 cells has been calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Correction for light absorption in fluorescence studies of protein-ligand interactions.

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.

Ligands↗

PubMed1983

Nuclear magnetic resonance evidence for a flexible region at the C-terminus of alpha-tropomyosin.

The 1H nuclear magnetic resonance spectrum of alpha-tropomyosin contains a number of sharp peaks indicative of the presence of small regions of high flexibility in the molecule. Removal of 9 to 11 residues from the C-terminus by digestion with carboxypeptidase A causes a marked decline in the intensity of these peaks. The difference is consistent with at least the C-terminal four residues of the sequence (-Met-Thr-Ser-Ile) being highly mobile. The conformation of the C-terminus is thus radically different from the alpha-helical coiled-coil from which the bulk of the molecule is constructed.

Carboxypeptidases↗

PubMed1983

Molecular flexibility in microtubule proteins: proton nuclear magnetic resonance characterization.

Bovine microtubule protein preparations have been examined by proton nuclear magnetic resonance (1H NMR) spectroscopy at 270 MHz. Sharp resonances have been identified as deriving from microtubule-associated proteins. These resonances persist after self-assembly of microtubule protein. Brief tryptic treatment of assembled microtubules, specifically cleaving the microtubule-associated protein HMW2 (Mr = 270 000), releases the pendant portion of HMW2 (Mr = 240 000), three-quarters of which is in a flexible conformation. Isolated tau protein and HMW2 protein both show substantial flexibility; on recombination with tubulin dimer, tau shows considerable decrease in flexibility whereas HMW2 is unaffected. The observations may have important implications for the interactions between microtubules and other cytoskeletal structures.

Amino Acids↗