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At least 19 recordsLinked to original sources
PubMed1992

Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase.

The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗
PubMed1986

Localization of noncovalently bound ethidium in free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer.

Ethidium binds tRNAfMet with 17-fold enhancement in the emission intensity at 600 nm. Fluorescence titration of tRNAfMet with ethidium indicates a single high-affinity site in tRNAfMet with a dissociation constant of 5 microM. Ethidium is apparently rigidly bound to tRNAfMet and effectively shielded from solvent. tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) with fluorophores at thiouridine, the 3'-terminus, and dihydrouridine, respectively, are prepared, and the singlet-singlet energy-transfer efficiencies between these fluorophores and noncovalently bound ethidium are determined. The transfer efficiency between bound ethidium and the fluorophore in tRNAfMet(8-13) determined by donor quenching and sensitized emission is the same, strongly suggesting that there is only one bound ethidium per tRNAfMet molecule. The apparent distances between ethidium and various fluorophores including 3'-fluorescein, the 8-13 photo-cross-link, and D-proflavin are 41, 19, and 30 A, respectively, assuming random orientation between the donor and the acceptor. The results suggest that noncovalently bound ethidium is intercalated in the amino acid acceptor stem. In the complex of tRNAfMet and methionyl-tRNA synthetase, the transfer efficiencies for the tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) are reduced, enhanced, and little changed, respectively. These methionyl-tRNA synthetase induced changes suggest changes in the conformation of the 3'-terminal unpaired bases and the relative orientation or location between tRNAfMet and ethidium upon binding of methionyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet.

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1970

Studies of transfer RNA tertiary structure by singlet-singlet energy transfer.

This distance between a base next to the anticodon of tRNA and the 3' CpCpA terminus of the molecule has been estimated by singlet-singlet energy transfer experiments. The energy donor was the Y base of unknown structure found in yeast tRNA(phe). Three different energy acceptors were used: acriflavine, proflavinyl acetic acid hydrazide, and 9-hydrazino acridine. These were attached to the periodate-oxidized 3' end of the tRNA. R(0)'s between 24 and 30 A were calculated for the three chromophore couples by assuming that the relative orientation of donor and acceptor is random. This assumption is supported by the consistency of the experimental results with all three acceptors and by studies of the fluorescence depolarization of Y. The energy transfer observed both by quenching of Y and enhanced activation of the acceptors is quite small, indicating that the anticodon is more than 40 A away from the amino acid accepting terminus. This places severe restrictions on the type of tertiary structure possible for tRNA.

Acridines↗
PubMed1974

Studies of transfer RNA tertiary structure of singlet-singlet energy transfer.

Five species of tRNAs bearing two different fluorescent groups were synthesized. These were suitable for intramolecular distance measurements by singlet-singlet energy transfer. The efficiency of energy transfer was determined from the sensitized emission of the energy acceptor. With the assumption that the relative orientation of donor and acceptor is random, the apparent distances between the 5'-end and the 3'-end, the 4-thiouridine and the 3'-end, the pseudouridine and 3'-end, the pseudouridine and the dihydrouridine in Escherichia coli formyl methionine tRNA, and between the 2-thiouridine (in the anticodon) and the 3'-end in E. coli glutamate tRNA were calculated to be 24 A, 38 A, 55 A, 36 A, and >65 A, respectively.

Chemical Phenomena↗
PubMed1976

Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes.

The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.

Binding Sites↗
PubMedDate not supplied

Studies of the dimensions of oligopeptides by singlet-singlet energy, transfer and theoretical calculations. I. Influence of glycine on the dimensions of tetrapeptides.

The efficiency of energy transfer between a fluorescent donor, L-tyrosine, and a fluorescent acceptor, L-tryptophan, has been determined in R'-L-Trp-L-Ala-L-Tyr-R", R-L-Trp-L-Ala-L-Ala-L-Tyr-R", and R'-L-Trp-Gly-L-Ala-L-Tyr-R" in ethanol solution. The protecting groups R' and R" were respectively tert-butyloxycarbonyl and methyl ester. A conformational theoretical analysis of molecules studied has been performed in parallel on the basis of semiempirical conformational potential energy function. In the theoretical models all the side chains have been represented by a methyl group. From the distribution of distances between chromophores obtained theoretically, transfer efficiencies have been computed assuming a random orientation of the chromophores (k-2 equals 2/3). The comparison of calculated efficiencies with the values determined experimentally for the same value of k-2 has been used as a check for the theoretical model. Both experimental and theoretical studies have been shown that the glycyl residue procudes a reduction of dimensions when it replaces in a tetrapeptide a residue with a beta-carbon atom such as the L-alanyl residue. However, only a qualitative agreement between experimental and theoretical values of the efficiencies has been obtained.

Alanine↗
PubMed1990

Singlet-singlet energy transfer along the helix of a double-stranded nucleic acid at room temperature.

An irreversible electronic energy trap has been formed in calf thymus DNA by methylating about 75% of its G bases at position N-7. This has allowed us to measure for the first time the efficiency of transfer of energy along the helix of a double-stranded nucleic acid at room temperature. It is found that about one out of every three photons absorbed by the other bases is trapped. We have also simulated the data with a stochastic model that uses the dipole-dipole interaction to calculate the efficiency of transfer. In order to approximate the experimental results, the model requires that: (i) the fluorescence quantum yield of T, C, and G in DNA be about 2 x 10(-3), which is about two orders of magnitude larger than the value of the fluorescence quantum yield reported for DNA; and (ii) the fluorescence quantum yield of A in DNA be negligibly small. Requirement (i) is consistent with energy transfer taking place before a very efficient fluorescence quenching process sets in, which could be formation of excited-state complexes (excimers) that do not fluoresce appreciably. Requirement (ii) implies a very short fluorescence lifetime for A, which is consistent with the reported absence of a significant number of photoproducts formed by A in DNA. The simulations find that, on the average, the excitation energy takes about 1.2 steps to reach the trap; that is to say, bases that are nearest and next nearest neighbors of the trap are, in effect, the only energy donors. Both intra- as well as interstrand energy transfer (the latter only for the C-trap base pair) make significant contributions. The value of the efficiency for pairwise base-base intrastrand transfer is about 60%, whereas those for base-trap intra- and interstand transfer are 90% and 80%, respectively. The corresponding values for the rate constant of transfer are 2 x 10(11), 1 x 10(12), and 4 x 10(11) s-1. Transfer is inefficient when A is the donor or the acceptor. In addition to the dipole-dipole term, the only other significant term in the expansion of the interaction potential is the dipole-quadrupole term which, however, makes only a small contribution to the overall transfer efficiency. The electron exchange interaction appears to be much less efficient than the coulombic interaction.

Animals↗
PubMed1986

Localization of L11 on the Escherichia coli ribosome by singlet-singlet energy transfer.

Isolated Escherichia coli ribosomal protein L11 was labeled with maleimidyl derivatives of coumarin or fluorescein at the thiol group of its single cysteine, then reconstituted singly or in pairs with other fluorescently labeled ribosomal components. The characteristics of fluorescence from the labeled protein were studied and its distance to other components was determined by non-radiative energy transfer. The distance between probes on L11 and cysteine residues on other proteins or the 3' end of the ribosomal RNAs were found to be: S1, 7.4-8.3 nm; S21, 7.6 nm; 23S RNA, 6.9 nm; 5S RNA, 7.6 nm; 16S RNA, greater than 8.5 nm. Considered together with previously published results these distances indicate that the location of L11 in the 50S subunit is below the lateral protuberance characterized by L7/L12.

Bacterial Proteins↗