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T Pan

Publications and source records attributed to T Pan.

At least 109 records · Page 6Linked to original sources
PubMed1991

Sequential assignments of the 1H NMR resonances of Zn(II)2 and 113Cd(II)2 derivatives of the DNA-binding domain of the GAL4 transcription factor reveal a novel structural motif for specific DNA recognition.

The DNA-binding domain of the GAL4 transcription factor, consisting of the 62 N-terminal amino acid residues and denoted GAL4(62*), contains a novel Zn(II)2Cys6 or Cd(II)2Cys6 binuclear cluster [Pan, T., & Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Specific DNA recognition requires residues located within as well as C terminal to this binuclear cluster. 1H NMR sequential assignments have been carried out on Zn(II)2- and 113Cd(II)2GAL4(62*) by using DQF-COSY, relayed COSY, double-relayed COSY, and NOESY. The ligands of the two tetrahedral metal-binding sites have been identified as Cys11, Cys14, Cys21, and Cys31 to one metal ion and Cys28, Cys38, Cys21, and Cys31 to the other metal ion with Cys21 and Cys31 as ligands shared between the two metal ions. No alpha-helices can be found within the GAL4(62*) structure, which consists of a series of turns to accommodate the metal cluster, followed by irregular loops and turns from residues 42 to 60, the "specificity region", whose sequence contributes importantly to specific DNA recognition. Long-distance NOE's are observed between residues forming the binuclear cluster and several residues within the specificity region, indicating that the latter is folded compactly onto the metal cluster. The requirement of the Zn(II)2Cys6 binuclear cluster and the specificity region for binding to DNA reveals GAL4 as a member of a class of specific DNA-binding proteins using a new structural motif for the recognition of specific DNA sequences. Specific DNA binding by this class of proteins is achieved by use of turns and loops that enclose a Zn(II)2Cys6 binuclear cluster, instead of alpha-helices or beta-strands as observed in specific DNA-binding proteins described previously.

Amino Acid Sequence↗
PubMed1990

The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster.

The DNA binding domain of the transcription factor LAC9 contains 6 cysteine residues with spacing in the primary peptide sequence identical to that found in the DNA binding domain of the GAL4 transcription factor. In GAL4, the CysX2CysX6CysX6CysX2CysX6Cys motif has been shown to form a Zn(II)2Cys6 binuclear cluster (Pan, T. and Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2077-2081), representing a new structure for a Zn(II)-containing transcription factor which differs from the "zinc finger" motif first described for TFIIIA. LAC9 has been shown to bind two Zn(II) ions (Halvorsen, Y. C., Nandabalan, K., and Dickson, R. D. (1990) J. Biol. Chem. 265, 13283-13289). The similarity of the amino acid sequence and the Cys spacing within the DNA binding domain suggest that LAC9 should also be capable of forming the Zn(II)2Cys6 cluster found in GAL4. A fragment of LAC9 consisting of 144 amino acid residues spanning the DNA binding domain has been prepared with 113Cd(II) substituted for the two native Zn(II) ions. 113Cd NMR of this fragment (denoted LAC9(85-228*] has been carried out in an attempt to test the hypothesis that LAC9, like GAL4, forms a binuclear cluster. The chemical shifts of the two bound 113Cd(II) ions, 705 and 692 ppm respectively, are consistent with ligation of each 113Cd(II) ion to 4 sulfur atoms. The best model for such ligation is that two of the cysteine S- form bridges between the two Cd(II) ions. Formation of a Zn(II)-Cd(II) hybrid form of LAC9(85-228*) has also been observed. We conclude that LAC9 contains a Zn(II)2Cys6 binuclear cluster as previously reported for GAL4.

Cations, Divalent↗
PubMed1990

Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor.

The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexpressed and purified from Escherichia coli previously. This fragment has been shown to contain 2.3 +/- 0.2 mol of Zn(II) per mole of protein [Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., & Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR(440-525)] with 113Cd(II) substituted for Zn(II) identifies 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus. The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands. Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear. Addition of EDTA to the protein restores the original two signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

The DNA binding domain of GAL4 forms a binuclear metal ion complex.

The transcription factor GAL4 from Saccharomyces cerevisiae requires Zn(II) or Cd(II) for specific recognition of the UASG sequence (Pan & Coleman, 1989). An N-terminal fragment consisting of the first 63 amino acid residues of GAL4 [GAL4(63)] has been obtained by partial tryptic proteolysis of a cloned and overproduced N-terminal domain of 149 residues, GAL(149). We show that GAL4(63) contains the minimal GAL4 DNA binding domain. GAL4(63) binds tightly 1-2 mol of Zn(II) or 2 mol of Cd(II). 113Cd NMR of 113Cd(II)-substituted GAL4(63) reveals structural identity between the metal binding domains of GAL4(63) and that of the larger precursor GAL4(149). 113Cd(II) can be substituted for the Zn(II) in GAL4(63), and two 113Cd NMR signals are observed at 706 and 669 ppm, both suggesting coordination of 113Cd(II) to three or four -S- ligands. With the exception of the N-terminal methionine, the only sulfur-containing residues are the six highly conserved cysteines. High-resolution 1H NMR of Zn(II)-GAL4(63) and Cd(II)-GAL4(63) show the two proteins to have almost identical conformations and to be present as monomers in solutions up to millimolar concentration. This leads us to postulate that GAL4 does not possess a TFIIIA-like "Zn-finger" but forms a binuclear metal cluster involving all six cysteines in a "cloverleaf"-like array. GAL4(63) contains about 60% alpha-helix, estimated from circular dichroism. Removal of the native Zn(II) causes substantial unfolding of the secondary structure. Unlike GAL4(149), the resultant apoprotein is not induced to refold by readdition of Zn(II) at low concentrations.

Amino Acid Sequence↗
PubMed1990

GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster.

The DNA-binding domain of the transcription factor GAL4, consisting of the 62 N-terminal residues and denoted GAL4(62*), contains a Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys motif, which has been shown previously to bind two Zn(II) or Cd(II) ions. Binding of Zn(II) or Cd(II) is essential for the recognition by GAL4 of the specific palindromic DNA sequence to which it binds upstream of genes for galactose-metabolizing enzymes, the UASG sequence. On the basis of the 113Cd NMR chemical shifts of the two bound 113Cd(II) ions, we propose a binuclear cluster model for this Zn(II)-binding subdomain. 1H-113Cd heteronuclear multiple-quantum NMR spectroscopy and phase-sensitive double-quantum filtered 1H correlation spectroscopy of the 112Cd(II)- and 113Cd(II)-substituted GAL4(62*) derivatives provide direct evidence that the two bound 113Cd(II) ions are coordinated only by the six cysteine residues, two of which form bridging ligands between the two 113Cd(II) ions. The latter can be identified from the pattern of 1H-113Cd J coupling. Thus a binuclear metal ion cluster rather than a "zinc finger" is formed by the six cysteine residues of the GAL4 DNA-binding domain. This model can be directly applied to eight other fungal transcription factors which have been shown to contain similarly spaced Cys6 clusters. 1H NMR spectra of apo-GAL4(62*) suggest conformational fluctuation of the metal-binding subdomain upon removal of Zn(II) or Cd(II). Both Cd(II)2- and Zn(II)2-containing species of GAL4 can be formed, and the similar 1H NMR spectra suggest similar conformations.

Amino Acid Sequence↗
PubMed1989

p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39.

The RNA binding protein of 56 residues encoded by the extreme 3' region of the gag gene of Rauscher murine leukemia virus (MuLV) has been chemically synthesized by a solid-phase synthesis approach. Since the peptide contains a Cys26-X2-Cys29-X4-His34-X2-Cys39 sequence that is shared by all retroviral gag polyproteins which has been proposed to be a metal binding region, it was of considerable interest to examine the metal binding properties of the complete p10 protein. As postulated, p10 binds the metal ions Cd(II), Co(II), and Zn(II). The Co(II) protein shows a set of d-d absorption bands typical of a tetrahedral Co(II) complex at 695 (epsilon = 565 M-1 cm-1), 642 (epsilon = 655 M-1 cm-1), and 615 nm (epsilon = 510 M-1 cm-1) and two intense bands at 349 (epsilon = 2460 M-1 cm-1) and 314 nm (epsilon = 4240 M-1 cm-1) typical of Co(II)----(-)S- charge transfer. The ultraviolet absorption spectrum also indicates Cd(II) binding by the appearance of a Cd(II)----(-)S- charge-transfer band at 255 nm. The 113Cd NMR spectrum of 113Cd(II)-p10 reveals one signal at delta = 648 ppm. This chemical shift correlates well with that predicted for ligation of 113Cd(II) to three -S- from the three Cys residues of p10. The chemical shift of 113Cd(II)-p10 changes by only 4 ppm upon binding of d(pA)6, indicating that the chelate complex is little changed by oligonucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution.

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-Binding Proteins↗
PubMed1989

Comparison of cooperative and isolated site binding of T4 gene 32 protein to ssDNA by 1H NMR.

Deuteriation of all aromatic protons of gene 32 protein (g32P) from phage T4, followed by selective introduction of specific protons, has allowed the precise identification of the number and magnitude of the chemical shift changes induced in the aromatic protons when g32P binds noncooperatively or cooperatively to nucleotides. Signals from five Tyr residues are shifted by binding of g32P to d(pA)8 or d(pA)40-60; however, the change from noncooperative, d(pA)8, to cooperative, d(pA)40-60, binding causes significant increases in the magnitudes of the shifts for only two of these Tyr signals. These two Tyr residues may interact directly with the nucleotide bases, while the shifts associated with the other three Tyr may be due to conformational changes in g32P upon ssDNA binding. Similar conclusions can be drawn for two of the six Phe residues whose protons undergo shifts upon nucleotide binding. Observation of selected proton signals allows for the first time detection by 1H NMR of changes in the proton signals from two Trp residues upon nucleotide binding. The side chains of two Tyr, one or two Phe, and one Trp are probably directly involved in nucleotide base-protein interactions. As assayed by the signals from the H2 and H8 protons of adenine, the bases of a bound nucleotide are undergoing a fast chemical exchange in the noncooperative mode of binding, but shift to slow exchange upon assuming the cooperative mode of ssDNA interaction. When bound to a polynucleotide, the A domain of g32P (residues 254-301) becomes more mobile, as reflected in sharpening of the 1H NMR signals from the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1989

Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor.

The transcription factor GAL4 from Saccharomyces cerevisiae contains a "zinc-finger"-like motif, Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys, within its DNA-binding domain. A GAL4 fragment consisting of residues 1-147 plus two additional residues from the cloning vector [denoted GAL4(149*)] has been cloned and overexpressed in Escherichia coli. This fragment includes the entire DNA-binding domain (residues 1-74). The homogeneous GAL4-(149*) protein contains 1-1.5 moles of Zn(II) per mole of protein. The GAL4(149*) protein binds tightly to the specific 17-base-pair palindromic DNA sequence found at GAL4 binding sites as shown by gel-retention assays using a 32P-labeled 23-mer containing this sequence. Removal of the intrinsic Zn(II) by EDTA at low pH abolishes binding to the 23-mer. The GAL4(149*) apoprotein can be reconstituted with Zn(II), Cd(II), or Co(II) with restoration of specific DNA binding. Titration of GAL4(149*) apoprotein with 113Cd(II) shows two 113Cd(II) binding sites on the molecule, one with delta of 707 ppm, suggesting coordination to four sulfur atoms, and one with delta of 669 ppm, suggesting coordination to three or four sulfur atoms. Because GAL4(149*) protein contains only six cysteine residues within its DNA-binding domain, the precise coordination of the two Cd(II) ions cannot be stated with certainty; one or more shared -S- ligands could exist. GAL4(149*) protein contains approximately 40% alpha-helix and approximately 20% beta-sheet, estimated from circular dichroism. Removal of the native Zn(II) ion causes limited unfolding of secondary structure, but less than one turn of alpha-helix. The binding of Zn(II), Cd(II), and, to a lesser extent, Co(II) to GAL4(149*) apoprotein protects the protein from proteolysis by trypsin, which produces a 13-kDa DNA-binding core.

Amino Acid Sequence↗
PubMed1981

Sensitive radioimmunoassay for vancomycin.

A radioimmunoassay for vancomycin has been developed which uses rabbit antiserum induced by vancomycin-bovine serum albumin conjugates and vancomycin labeled with 3H or 125I. Using either isotope, the method is simple and reproducible and has a sensitivity of 4 or 0.04 ng/ml, depending on the tracer used. This is 200- to 20,000-fold improvement in sensitivity compared with the most sensitive bioassay. Drug levels in serum or urine samples from patients receiving vancomycin can be determined by this assay procedure without processing. The data obtained with 3H and 125I labels were in good agreement. Patients' plasma vancomycin concentrations determined by radioimmunoassay correlated well with those determined by bioassay when the drug was administered intravenously. However, after oral administration the drug could be detected only by radioimmunoassay. The antiserum was evaluated for cross-reactivity with a wide variety of antibiotics and cancer chemotherapeutic agents, and no significant interference was found.

Animals↗
PubMed1980

New schedule for tobramycin administration.

Ten patients received a loading dose of tobramycin of 60 mg/m2 intravenously over 0.5 h followed immediately by 60 mg/m2 over 2 h every 4 h. The highest mean serum concentration (at the end of the loading dose) was 6.0 +/- 0.3 microgram/ml (range, 3.2 to 10.9 microgram/ml). The mean serum concentration 2 h after the end of the initial 2-h infusion was 3.0 +/- 0.2 microgram/ml (range, 1.6 to 3.9 microgram/ml). This schedule of tobramycin was used to treat 117 patients with presumed or proven infection. There were no differences in the mean serum concentrations of tobramycin at comparable times on days 3 to 4 and 6 to 7. Only 60% of patients had serum levels between 3 micrograms/ml (trough) and 10 micrograms/ml (peak). This intermittent schedule of administration resulted in substantial fluctuations in serum concentrations of tobramycin and produced trough concentrations which were too low or peak concentrations which were too high in some patients.

Adolescent↗
PubMed1978

PC-904, a new semisynthetic penicillin.

Sodium 6{d(-)-alpha(4-hydroxyl-1,5 naphthyridine-3-carboxamido)phenylacetamido} penicillanate (PC-904) is a new semisynthetic penicillin with broad-spectrum activity against gram-positive cocci and gram-negative bacilli. At a concentration of 1.56 mug/ml, it inhibited 100% of isolates of Proteus mirabilis, 89% of Pseudomonas aeruginosa, 67% of Escherichia coli, and 45% of Enterobacter spp. At a concentration of 12.5 mug/ml, it inhibited 75% of Klebsiella spp. and 67% of Serratia marcescens. PC-904 failed to inhibit the growth of gram-negative bacilli when large inocula were used. Some differences were noted when organisms were tested in different media or at different hydrogen ion concentrations. It is more active than mezlocillin, azlocillin, ticarcillin, carbenicillin, and amoxicillin against E. coli, Klebsiella spp., and P. aeruginosa.

Ampicillin↗
PubMed1978

Racial influences in open-angle glaucoma.

Blindness due to glaucoma is much greater in blacks than in whites. Using a computerized diagnostic index we identified more than 1,300 patients being treated for glaucoma in our outpatient clinics. Analysis of this clinic population indicated that open-angle glaucoma is more prevalent among blacks than in whites. They also indicate that open-angle glaucoma occurs at a younger age in the black population. We have very limited data that tend to indicate that open-angle glaucoma is a more severe disease in blacks than in whites; however, this evidence is far from conclusive. Finally, our findings indicate that in our institution we are seeing the full range of the glaucomatous process in the black population and not merely the end-stage disease.

Adult↗
PubMed1977

Mezlocillin: in vitro studies of a new broad-spectrum penicillin.

Mezlocillin is a new semisynthetic penicillin that inhibited 71% of the isolates of Serratia marcescens, 67% of Escherichia coli, 50% of Enterobacter spp., and 49% of Klebsiella spp. at a concentration of 12.5 mug/ml. It is also active against both indole-positive and -negative Proteus spp. and gram-positive cocci, except penicillin G-resistant Staphylococcus aureus. At a concentration of 100 mug/ml, it inhibited 94% of the isolates of Pseudomonas aeruginosa. It is more active than ampicillin, carbenicillin, and cephalothin against some gram-negative bacilli.

Bacteria↗
PubMed1977

A new pencillin with anti-klebsiella activity: 3-(5-tetrazolyl) penam.

The in vitro activity of a new semi-synthetic penicillin, CP-35,587, 3-(5-tetrazolyl) penam, was investigated against 496 clinical isolates of gram-negative bacilli and 113 clinical isolates of gram-positive cocci. All of the gram-positive cocci were sensitive to CP-35,587 except penicillin G resistant isolates of Staphylococcus aureus. This antibiotic inhibited a majority of isolates of Escherichia coli, Klebsiella spp. and Proteus mirabilis at a concentration of 6.25 microgram/ml. Also, approximately half of the isolates of Serratia marcescens and Enterobacter spp. were inhibited at a concentration of 12.5 microgram/ml. CP-35,587 was inactive when high concentrations of organisms were used as inocula. CP-35,587 was more active than mezlocillin, azlocillin, amoxicillin, ticarcillin and carbenicillin against isolates of K. pneumoniae, but other penicillins were more acte than CP-35,587 against other species of gram-negative bacilli.

Bacteria↗
PubMed1976

Effect of cephalothin on growth patterns of micro-organisms.

Isolates of Staphylococcus aureus, Escherichia coli. Klebsiella pneumoniae and Proteus mirabilis were incubated in the presence of inhibitory concentrations of cephalothin. After destruction of the antibiotic, there was a lag phase before S. aureus began to proliferate again. When similar experiments were conducted with E. coli, K. pneumoniae, and P. mirabilis, no lag phase was observed. This data suggests that the inhibitory activity of cephalosporins may be different for gram-positive cocci and gram-negative bacilli.

Bacteria↗