Biomedical subjects

Robert E London

Publications and source records attributed to Robert E London.

At least 19 recordsLinked to original sources

PubMed2006

Photoactivated h/d exchange in tyrosine: involvement of a radical anion intermediate.

The aromatic hydrogen nuclei of tyrosine are photochemically labile and exchange with deuterons in neutral D(2)O solution. The site meta to the ring hydroxyl substituent is preferentially deuterated, exhibiting a meta/ortho deuteration rate of approximately 4:1. In contrast with acid-catalyzed H/D exchange and with nearly all of the reported photoactivated H/D exchange studies, the UV-induced H/D exchange of tyrosine is optimal at pH 9 and is effectively quenched at acid pH. Photochemical H/D exchange is strongly stimulated by the alpha-amino group (the aromatic hydrogens of p-cresol are far less subject to exchange) and by imidazole or phosphate buffers. On the basis of the results obtained here and on the previously identified cyclohexadienyl radical (Bussandri, A.; van Willigen, H. J. Phys. Chem. A 2002, 106, 1524-1532), we conclude that the exchange reaction involves a radical intermediate and results from two distinct roles of tyrosine: (1) as a phototransducer of light energy into solvated electrons (e(aq)(-)), and (2) as an acceptor of an electron to create a radical anion intermediate which is rapidly protonated, yielding a neutral cyclohexadienyl radical. Regeneration of the tyrosine can occur via a bimolecular redox reaction of the cyclohexadienyl and phenoxyl radicals to yield a carbocation/phenoxide pair, followed by deprotonation of the carbocation. The oxidation step is pH dependent, requiring the deprotonated form of the cyclohexadienyl radical. The H/D exchange thus results from a cyclic one-electron (Birch) reduction/protonation/reoxidation (by phenoxyl radical)/deprotonation cycle. Consistent with these mechanistic conclusions, the aromatic hydrogens of tyrosine O-methyl ether are photochemically inert, but become labile in the presence of tyrosine at high pH. The deuteration rate of O-methyl tyrosine is lower than that of tyrosine and shows a preference for the ortho positions. This difference is proposed to result from a variation in the oxidation step, characterized by a preferential oxidation of a cyclohexadienyl resonance structure with the unpaired electron localized on the oxygen substituent.

Amino Acids↗

PubMed2006

NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general increase in the Hill coefficients observed in the complex is consistent with the screening of the interacting lysine residues by the DNA. The pKa of K312 residue increased to 10.58 in the complex, probably due to salt bridge formation with the 5'-phosphate group of the DNA. The pKa values obtained for the lyase domain of Pol lambda in the present study are consistent with recent crystallographic studies of Pol beta complexed with 5-phosphorylated abasic sugar analogues in nicked DNA which reveal an open site with no obvious interactions that would significantly depress the pK value for the active site lysine residue. It is suggested that due to the heterogeneity of the damaged DNA substrates with which Pol lambda as well as other related polymerases may be required to bind, the unexpectedly poor optimization of the lyase catalytic site may reflect a compromise of flexibility with catalytic efficiency.

Binding Sites↗

PubMed2005

A thymine isostere in the templating position disrupts assembly of the closed DNA polymerase beta ternary complex.

The high fidelity of the DNA polymerization process is critically important for the stability of the cellular genome. The role of template and incoming nucleotide base pairing in polymerase fidelity has recently been explored by the use of nucleotide isosteres, which preserve the steric but not the electronic properties of the corresponding bases. The DNA repair enzyme, DNA polymerase beta (Pol beta), is among the most discriminating, being inactive when the thymine isostere difluorotoluene (DFT) is present in the templating base position. To explore the physical basis for this inactivity, we have performed NMR studies on [methyl-13C]methionine-labeled Pol beta complexed with double-hairpin DNA, used to model the gapped nucleotide substrate, and having either a thymine or a DFT isostere at the templating base position. The six methionine residues distributed throughout the enzyme provide useful conformational probes of the lyase and polymerase domains and subdomains. Analysis of the proton shift of Met282 that results from formation of an abortive Pol beta-gapped DNA-dATP complex is consistent with an open to closed conformational change of the enzyme predicted from crystal structures. In contrast, the same resonance is nearly unshifted when a ternary complex is formed from dATP and gapped DNA in which a DFT isostere replaces thymine at the templating base position. Alternatively, the resonances of Met191 and Met155, located in the catalytic subdomain, show perturbations upon formation of the abortive ternary complex, which are qualitatively similar, but significantly weaker, than the changes observed when thymine is present at the templating base position. The changes in the Met155 and Met191 methyl resonances are in fact more similar to those observed in the binary Pol beta-dATP complex. These studies demonstrate that the block in catalysis is directly related to the absence of the set of conformational transitions that include the "open" to "closed" transition monitored by Met282.

Animals↗

PubMed2005

Structure-function studies of DNA polymerase lambda.

DNA polymerase lambda is a member of the X family of polymerases that is implicated in non-homologous end-joining of double-strand breaks in DNA and in base excision repair of DNA damage. To better understand the roles of DNA polymerase lambda in these repair pathways, here we review its structure and biochemical properties, with emphasis on its gap-filling polymerization activity, its dRP lyase activity and its unusual DNA synthetic (in)fidelity.

DNA Polymerase beta↗

PubMed2005

Calorimetric studies of ligand binding in R67 dihydrofolate reductase.

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.

Calorimetry↗

PubMed2005

Structural insights into the mechanism of nuclease A, a betabeta alpha metal nuclease from Anabaena.

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the delta(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a betabeta alpha metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites approximately 26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the betabeta alpha metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

Amino Acid Motifs↗

PubMed2005

Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury.

Under hypercontractile conditions associated with increased intracellular calcium, male hearts show enhanced ischemia/reperfusion injury compared to female hearts. Our aim in this study was to identify the specific estrogen receptor involved in this gender difference. Following brief treatment with isoproterenol, isolated mouse hearts were subjected to ischemia and reperfusion. Postischemic contractile function and infarct size were measured in wild-type (WT) male and female hearts, and female hearts lacking functional alpha estrogen receptor (alpha ERKO), or the beta estrogen receptor (beta ERKO). WT male hearts exhibited significantly less functional recovery and more necrosis than WT females. alpha ERKO female hearts exhibited ischemia/reperfusion injury similar to that observed in WT females, whereas beta ERKO females exhibited significantly less functional recovery than WT females and were similar to WT males. These data suggest that estrogen, through the beta-estrogen receptor, plays a role in the protection observed in the female heart. Furthermore, we identified genes that were differentially expressed in beta ERKO female hearts compared to alpha ERKO and WT female hearts, and found altered expression of a number of metabolism genes, which may be important in ischemic injury. We further showed that WT female hearts have increased ratio of carbohydrate to fatty acid metabolism relative to WT males.

Animals↗

PubMed2005

Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit.

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.

Alcohols↗

PubMed2004

Backbone dynamics of the RNase H domain of HIV-1 reverse transcriptase.

Previous NMR relaxation studies of the isolated RNase H domain of HIV-1 reverse transcriptase at low pH have revealed that it is substantially more dynamic and less ordered than the relatively stable and catalytically active E. coli RNase HI. Using more recently developed techniques, we have investigated the dynamic behavior of the RNase H domain of HIV-1 reverse transcriptase at a more physiological pH (6.8), under a variety of solution conditions: no Mg(2+), 80 mM Mg(2+), and 80 mM Mg(2+) plus AMP ligand. In addition, we have repeated the previous measurements on a sample containing 100 mM sodium acetate, pH 5.4. Under all conditions studied, the order parameters from NMR relaxation analysis are uniformly high (>0.8) for most of the domain with the exception of the C-terminal region. Subtle differences can be found among the conditions studied, although the statistical significance of the differences is marginal. Residues 71-114 show a slight increase in order parameter with the addition of 5'-AMP. Conformational exchange, measured with CPMG relaxation dispersion experiments in the presence of Mg and AMP, were detected for some NH sites, predominantly located in the N-terminal region of the protein near strands beta2 and beta3 and helix alpha(A) (residues 28-69). In contrast with earlier studies indicating pathologically extreme dynamic behavior that apparently correlated with inactivity of the isolated domain, the relaxation analysis under the conditions of the present study yielded parameters that are more similar to those of the active E. coli RNase HI. A comparison of the order parameters obtained from a model-free analysis of the relaxation data with the B-factors in the crystal structures of the RNase H domain, both for the isolated domain and for the full HIV-1 reverse transcriptase structure, suggests that the dynamic behavior is similar in all cases.

HIV Reverse Transcriptase↗

PubMed2004

Dynamic characterization of a DNA repair enzyme: NMR studies of [methyl-13C]methionine-labeled DNA polymerase beta.

Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.

Animals↗

PubMed2004

X-ray and NMR characterization of covalent complexes of trypsin, borate, and alcohols.

An understanding of the physiological and toxicological properties of borate and the utilization of boronic acids in drug development require a basic understanding of borate-enzyme chemistry. We report here the extension of our recent NMR studies indicating the formation of a ternary borate-alcohol-trypsin complex. Crystallographic and solution state NMR studies of porcine trypsin were performed in the presence of borate and either of three alcohols designed to bind to the S1 affinity subsite: 4-aminobutanol, guanidine-3-propanol, and 4-hydroxymethylbenzamidine. Quaternary complexes of trypsin, borate, S1-binding alcohol, and ethylene glycol (a cryoprotectant), as well as a ternary trypsin, borate, and ethylene glycol complex have been observed in the crystalline state. Borate forms ester bonds to Ser195, ethylene glycol (two bonds), and the S1-binding alcohol (if present). Spectra from (1)H and (11)B NMR studies confirm that these complexes also exist in solution and also provide evidence for the formation of ternary trypsin, borate, and S1-subsite alcohol complexes which are not observed in the crystals using our experimental protocols. Analysis of eight crystal structures indicates that formation of an active site borate complex is in all cases accompanied by a significant (approximately 4%) increase in the b-axis dimension of the unit cell. Presumably, our inability to observe the ternary complexes in the crystalline state arises from the lower stability of these complexes and consequent inability to overcome the constraints imposed by the lattice contacts. A mechanism for the coupling of the lattice contacts with the active site that involves a conformational rearrangement of Gln192 is suggested. The structures presented here represent the first crystallographic demonstration of covalent binding of an enzyme by borate.

Amino Alcohols↗

PubMed2004

NvAssign: protein NMR spectral assignment with NMRView.

MOTIVATION: Nuclear magnetic resonance (NMR) protein studies rely on the accurate assignment of resonances. The general procedure is to (1) pick peaks, (2) cluster data from various experiments or spectra, (3) assign peaks to the sequence and (4) verify the assignments with the spectra. Many algorithms already exist for automating the assignment process (step 3). What is lacking is a flexible interface to help a spectroscopist easily move from clustering (step 2) to assignment algorithms (step 3) and back to verification of the algorithm output with spectral analysis (step 4). RESULTS: A software module, NvAssign, was written for use with NMRView. It is a significant extension of the previous CBCA module. The module provides a flexible interface to cluster data and interact with the existing assignment algorithms. Further, the software module is able to read the results of other algorithms so that the data can be easily verified by spectral analysis. The generalized interface is demonstrated by connecting the clustered data with the assignment algorithms PACES and MONTE using previously assigned data for the lyase domain of DNA polymerase lambda. The spectral analysis program NMRView is now able to read the output of these programs for simplified analysis and verification. AVAILABILITY: NvAssign is available from http://dir.niehs.nih.gov/dirnmr/nvassign

Algorithms↗

PubMed2004

Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III.

DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.

Amino Acid Sequence↗

PubMed2004

Male/female differences in intracellular Na+ regulation during ischemia/reperfusion in mouse heart.

We previously showed that beta-adrenergic stimulation revealed male/female differences in susceptibility to ischemia/reperfusion (I/R) injury. To explore whether altered [Na(+)](i) regulation is involved in the mechanism of this sex difference, we measured [Na(+)](i) by (23)Na NMR spectroscopy in isolated perfused mouse hearts. [Na(+)](i) increased to 195 +/- 3% (mean +/- S.E.) of the pre-ischemic level at 20 min of ischemia in male hearts, whereas [Na(+)](i) accumulation was slightly less in female hearts (176 +/- 2%, P < 0.05). There was no significant difference in the recovery of contractile function after reperfusion (male: 30.6 +/- 3.8%; female: 35.0 +/- 1.9%; P > 0.05). If hearts were treated with isoproterenol (ISO, 10 nmol/l), males exhibited significantly poorer recovery of post-ischemic contractile function than females (male: 13.0 +/- 1.9%; female: 28.1 +/- 1.2%; P < 0.05), and a significantly higher [Na(+)](i) accumulation during ischemia (male: 218 +/- 8%; female: 171 +/- 2%; P < 0.05). This ISO-induced male/female difference in [Na(+)](i) accumulation or contractile function was blocked by the nitric oxide synthase inhibitor, N(omega)-nitro-l-arginine methyl ester (1 micromol/l). Furthermore, in ISO-treated hearts, the Na(+)/K(+)-ATPase inhibitor, ouabain (200 micromol/l) did not abolish the male/female difference in [Na(+)](i) accumulation during I/R or functional protection. Thus the data show that the sex difference in the [Na(+)](i) regulation is mediated through a NO-dependent mechanism, and the difference in susceptibility to I/R injury appears to result from a difference in Na(+) influx.

Animals↗

PubMed2003

NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal focal adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called focal adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.

Amino Acid Sequence↗

PubMed2003

NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity.

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.

Amides↗

PubMed2003

Gender differences in sarcoplasmic reticulum calcium loading after isoproterenol.

Males exhibit enhanced myocardial ischemia-reperfusion injury versus females under hypercontractile conditions associated with increased sarcoplasmic reticulum (SR) Ca2+. We therefore examined whether there were gender differences in SR Ca2+. We used NMR Ca2+ indicator 1,2-bis(2-amino-5,6-difluorophenoxy)-ethane-N,N,N',N'-tetraacetic acid to measure SR Ca2+ in perfused rabbit hearts. Isoproterenol increased SR Ca2+ in males from a baseline of 1.13 +/- 0.07 to 1.52 +/- 0.24 mM (P < 0.05). Female hearts had basal SR Ca2+ that was not significantly different from males (1.04 +/- 0.03 mM), and addition of isoproterenol to females resulted in a time-averaged SR Ca2+ (0.97 +/- 0.07 mM) that was significantly less than in males. To confirm this difference, we measured caffeine-induced release of SR Ca2+ with fura-2 in isolated ventricular myocytes. Ca2+ release after caffeine in untreated male myocytes was 377 +/- 41 nM and increased to 650 +/- 55 nM in isoproterenol-treated myocytes (P < 0.05). Ca2+ release after caffeine addition in untreated females was 376 +/- 27 nM and increased to 503 +/- 49 nM with isoproterenol, significantly less than in male myocytes treated with isoproterenol (P < 0.05). Treatment of female myocytes with NG-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS), resulted in higher SR Ca2+ release than that measured in females treated only with isoproterenol and was not significantly different from that measured in males with isoproterenol. Female myocytes also have significantly higher levels of neuronal NOS. This gender difference in SR Ca2+ handling may contribute to reduced ischemia-reperfusion injury observed in females.

Adrenergic beta-Agonists↗

PubMed2003

Solution structure of the lyase domain of human DNA polymerase lambda.

DNA polymerase lambda (pol lambda) is a recently discovered nuclear enzyme belonging to the pol X family of DNA polymerases that exhibits a 32% sequence identity with the nuclear DNA repair protein, pol beta. Structural modeling suggests that pol lambda contains the palm, fingers, thumb, and 8 kDa lyase domains present in pol beta, as well as an additional N-terminal BRCT domain and a serine-proline-rich linker that are presumably involved in protein-protein interactions. The 8 kDa domain of pol beta is important for DNA binding and contains the dRP lyase activity, which is the rate-limiting step in the single-nucleotide base excision repair (BER) pathway of damaged DNA. Recently, it was shown that the 8 kDa domain of pol lambda also contains the dRP lyase activity. To gain further insight into the catalytic mechanism of dRP removal by pol lambda, we have determined the solution structure of the 8 kDa lyase domain of human DNA pol lambda via multidimensional NMR methods and the ARIA program. The resulting structures exhibited a high degree of similarity with the 8 kDa lyase domain of pol beta. Specifically, the side chains of residues W274, R275, Y279, K307, R308, and K312 are in similar positions to the functionally important side chains of residues H34, K35, Y39, K60, K68, and K72 in the 8 kDa lyase domain of pol beta. This suggests that, on the basis of the proposed roles of these residues in pol beta, the corresponding pol lambda side chains may be involved in DNA binding and dRP lyase activity. The structural alignment of W274 (pol lambda) with H34 (pol beta) indicates that the former is probably involved in a similar base stacking interaction with template DNA at the position of the gap, in contrast with several previous proposals which aligned D272 with H34. In a few cases for which there is a nonconservative substitution in the sequence alignment, a structural comparison shows a positionally and, hence, probably a functionally equivalent residue, e.g., K60 in pol beta and K307 in pol lambda. Additionally, on the basis of the structural alignment obtained, several previously proposed mechanistic hypotheses can be evaluated.

Amino Acid Sequence↗