Optically detected zero field magnetic resonance characterization of a bromine atom-containing polynucleotide, poly(dA-br5dU).

Phosphorescence and optically detected zero field magnetic resonance ( ODMR ) spectra are reported for a bromine atom-containing polynucleotide, poly(dA- br5dU ). The triplet state luminescence of poly(dA- br5dU ) is dominated by the phosphorescence of the bromouracil base which possesses sub-millisecond triplet lifetimes. Characteristic multiple slow passage ODMR transitions, which are observed in both br5dUrd and poly(dA- br5dU ), are assigned to the triplet state of bromouracil. In addition, an abnormally-perturbed adenine triplet state, which is not apparent in the phosphorescence spectrum of poly(dA- br5dU ), is detected and identified by its slow passage ODMR and amplitude-modulated phosphorescence microwave double resonance spectra. It is proposed that the perturbed adenine is a minor component of the polynucleotide structure which is present in regions of altered stacking induced by the high polarizability of the Br atom.

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy.

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.

Binding of recA protein to single- and double-stranded polynucleotides occurs without involvement of its aromatic residues in stacking interactions with nucleotide bases.

Phosphorescence and optically detected triplet state magnetic resonance (ODMR) spectroscopy studies of recA protein and its complexes with poly(5-HgU) and poly(dA-5BrdU) show that the two tryptophan residues are not involved in stacking interactions with the nucleotide bases of either single- or double-stranded polynucleotides. Solvent conditions which induce preferential binding to single-stranded ligands result in a shortening of the tyrosine phosphorescence lifetime, which is further reduced upon binding to poly(5-HgU). This suggests a change in the global conformation or self-aggregation state of the protein. Binding to poly(dA-5BrdU) induces small changes in the tryptophan zero field splittings of recA, but significant changes on those of 5BrdU, which are consistent with recA binding to the minor groove of the polynucleotide.

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.

Study of complexes of a tryptophan-free mutant of E. coli trp aporepressor with tryptophan analogues using optically detected magnetic resonance (ODMR).

Phosphorescence and optically detected magnetic resonance (ODMR) spectra of tryptophan (W) and several of its analogues (4-, 5-, 6-methyltryptophan (MeW); 4-, 5-, 6-fluorotryptophan (FW); 5-bromotryptophan) are compared with those of complexes formed with the W-free trp aporepressor from Escherichia coli (W19,99F). W19,99F binds W and each analogue except 4-FW with an estimated KD < or = 30 microM; triplet state spectroscopic and kinetic effects that accompany binding at the corepressor site are reported. ODMR data for the MeW isomers are presented for the first time. No binding of 7-azaW is observed, in agreement with the low affinity found by previous workers.

alpha-Bungarotoxin binding to two acetylcholine receptor alpha-peptides and their methylmercury-modified analogs: intrinsic phosphorescence and optically detected magnetic resonance studies.

Phosphorescence and optically detected magnetic resonance (ODMR) have been used to characterize two synthetic peptides, alpha 181-198 and alpha 185-196, of the major binding determinant of the alpha-acetylcholine receptor (AChR) of Torpedo californica and its interaction with alpha-bungarotoxin (BgTX) using Trp as an intrinsic probe. BgTX conformational changes are suggested upon complexation with the peptides. Methylmercury-modified peptides show conformational heterogeneity which brings some of the modified Cys residues into proximity of peptide Trp(s). These modified peptides, when bound to BgTX, undergo structural changes which remove the tagged Cys from its close contact with the Trp residue(s) of the peptide.

Investigation of phospholipase-lipid interactions by optical detection of triplet state magnetic resonance spectroscopy.

We have investigated the binding of porcine pancreatic phospholipase A2 (PA2) to n-hexadecylphosphocholine (C16PN) micelles using optical detection of triplet state magnetic resonance (ODMR) spectroscopy. The zero field splittings (zfs) of the single Trp3 residue undergo significant changes upon binding of PA2 to C16PN micelles. Zfs titrations of PA2 vs C16PN indicate that the binding stoichiometry is C16PN:PA2 approximately 25. A reduction of the (E) parameter from 1.227 to 1.135 GHz is postulated to result from Stark effects caused by the binding of a polar group (possibly phosphocholine) near Trp3 in the PA2-C16PN micelle complex.

Comparative optically detected magnetic resonance studies of mammalian phospholipase A2-lipid interactions.

A major difference between porcine and bovine pancreatic phospholipase A2 (PA2) is the relatively low affinity of the bovine enzyme for lipid-water interfaces. We have investigated the binding of porcine, bovine, and equine PA2 to n-hexadecylphosphocholine (C16-PC) micelles using optically detected magnetic resonance (ODMR) spectroscopy. The zero field splittings (ZFS) of the single Trp-3 residue undergo significant changes upon binding of PA2 to C16-PC micelles. ZFS titrations of PA2 vs C16-PC indicate that porcine and equine enzymes have similar binding affinity and stoichiometry, while bovine PA2 binds much more weakly to the lipid-water interfaces. This may be attributed to the differences in the amino acid composition and the conformation of the binding sites for lipid-water interfaces of these enzymes.

Energy transfer in complexes of E. coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid).

The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.

Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E. coli SSB protein to single-stranded polynucleotides.

The binding of both wild-type and point-mutated E. coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60. This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine.

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides.

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.

Evidence for stacking interactions between 5-mercurated polyuridylic acid and HIV-1 p7 nucleocapsid protein obtained by phosphorescence and optically detected magnetic resonance (ODMR).

The photoexcited triplet state of Trp-37 in the C-terminal zinc finger of the HIV-1 p7 nucleocapsid protein was used as a probe of p7 interactions with the heavy atom-derivatized RNA homopolymer, poly-5-mercuriuridylic acid (5-HgU). Binding of p7 to 5-HgU (Hg blocked with 2-mercaptoethanol) produces an external heavy atom effect (HAE) on Trp-37 characterized by fluorescence quenching, reduction of the phosphorescence lifetime by three orders of magnitude, and the appearance of the D+E phosphorescence-detected ODMR signal, absent in unperturbed Trp, but induced by a HAE. The details of the HAE are consistent with out-of-plane van der Waals contact of Hg with the indole chromophore of Trp-37. Steric requirements suggest further that the Trp-RNA contact occurs via an aromatic stacking interaction.

Determination of relative triplet sublevel populating rates during optical pumping using ODMR.

A method is introduced, based on optical detection of triplet state magnetic resonance (ODMR), to determine the relative populating rates of photoexcited triplet state sublevels during optical pumping. Phosphorescence transients induced by microwave rapid passage during optical pumping are analyzed globally utilizing kinetic parameters obtained from separate microwave-induced delayed phosphorescence measurements to obtain relative sublevel populating rates. Results are unaffected by phosphorescence from triplet populations that do not yield an ODMR response. The method is applied to the triplet state of the indole chromophore in various environments to reveal the effects of local interactions on the pattern of intersystem crossing. Enhanced spin--orbit coupling effects are attributed to interactions that reduce the planar symmetry of the indole chromophore.

Conformer interconversion in the LC analysis of triostin A and its under-N-methylated synthetic analogue.

Multiple peak formation and interconversion in the liquid chromatographic (LC) analysis of triostin A and its under-N-methylated synthetic analogue, [N-MeCys3,N-MeCys7]-TANDEM (MCTANDEM), are investigated as a function of column temperature. Slow interconversion between chromatographic peaks, ascribed to the presence of the n- and p-solution conformers of the peptides, is exhibited in the normal-phase elution profiles of triostin A and in the reversed-phase elution profiles of MCTANDEM. A chromatographic model is developed to estimate the kinetics of conformer interconversion. Reversed-phase LC analysis of the n- and p-conformers of MCTANDEM yields a value of 0.01/s for the apparent interconversion rate constant (kn-p) at 25 degrees C, with a corresponding activation energy of 16 kcal/mol. Normal-phase LC analysis of the n- and p-conformer interconversion of triostin A dissolved in chloroform results in a value of 0.04/s for kn-p at 25 degrees C, with a corresponding activation energy of 18 kcal/mol. For triostin A, normal-phase LC findings as a function of column temperature are compared with 1H nuclear magnetic resonance (NMR) line-width measurements between 80 degrees C and 140 degrees C for the n- and p-conformers, which yield an activation energy of 19 kcal/mol and an extrapolated value of 0.02/s at 25 degrees C for kn-p in deuteriochloroform.

Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme.

The radiative quantum yield, phi op, of the triplet state of human alpha-lactalbumin (HLA) has been measured in the temperature range between 6 K and the softening point of the aqueous glass (approximately 150 K). phi op has little temperature dependence below approximately 30 K, but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-state model in which the phosphorescence originates primarily from a donor, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy transfer process. The model assumes that the acceptor (Trp 60) triplet state undergoes radiationless deactivation by a proximal disulfide residue, while the donor (Trp 104) has no such extrinsic quencher. The decrease of phi op with increasing temperature is accounted for by the thermally activated triplet-triplet energy transfer process. The disulfide quenching rate constant itself is assumed to be temperature independent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphorescence quenching in HLA occurs with an activation energy of 97 cm-1, which we associate with a barrier to the energy transfer process. The data are fit well by the model if we assume a value for the temperature-independent disulfide quenching constant of kQ > 3 s-1 that is consistent with recent measurements on indole-disulfide model systems (Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). Similar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calculations since it is the simplest, lacking Trp 26, a residue not considered in the model, that probably contributes significantly to the phosphorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPLA, which contains Trp 104 but lacks Trp 60, shows qualitatively less thermally induced phosphorescence quenching than HLA, BLA, and HEWL, thus supporting the postulated quenching model.

Optically detected magnetic resonance study of the interaction of an arsenic(III) derivative of cacodylic acid with EcoRI methyl transferase.

The interaction of the enzyme Escherichia coli RI methyl transferase (methylase) with an arsenic(III) derivative of cacodylic acid has been investigated by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy in zero applied magnetic field. The reactive derivative (CH3)2AsSR is formed by the reduction of cacodylate by a thiol. The As(III) derivative binds to the enzyme by mercaptide exchange with a cysteine (Cys) residue located close to a tryptophan (Trp) site. The arsenical binding selectively induces an external heavy-atom effect, perturbing the nearby Trp residue in the enzyme. Zero-field splittings (ZFS) and total decay rate constants of the individual triplet-state sublevels of the Trp residue in the presence and absence of perturbation by As(III) have been determined. The perturbed Trp shows a large reduction in the overall decay lifetime compared with unperturbed Trp residue, exhibiting a high selectively for the Tx sublevel. This selectivity suggests that the As atom lies in the xz plane of the principal magnetic axis system of Trp, but not directly along the z (out-of-plane) axis. The accessibility of this enzyme binding site to the arsenical is decreased upon forming a ternary complex of methylase with sinefungin and a DNA oligomer, d[GCGAA(BrU)(BrU)CGC], containing two 5-bromouracil (BrU) bases in place of thymine within the hexadeoxynucleotide recognition sequence. This result indicates that the arsenical binding site in methylase which produces the Trp heavy-atom effect is protected from this ligand by ternary complex formation or the enzyme undergoes a conformation change, removing the Cys from the Trp site. This protection is also observed in fluorescence quenching experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Methylmercury(II) binding to single-stranded and duplex DNA: complexes formed are distinguishable by optical detection of magnetic resonance spectroscopy.

Binding of CH3Hg(II) to duplex and single-stranded calf thymus DNA leads to an external heavy atom effect that is associated with the formation of complexes directly with the purine and pyrimidine bases. When CH3Hg(II) is added at a concentration insufficient to cause denaturation, clearly distinguishable optical detection of magnetic resonance spectra are observed from the duplex and single-stranded DNA complexes. Comparison of the dominant signals with those observed from CH3Hg(II) complexes of model mononucleotides and mononucleosides allows their identification as guanine complexed at N7 in the duplex sample and thymine complexed at N3 in the single-stranded sample. On the basis of these measurements, it is estimated that this experiment presently is capable of detecting about 2% single-stranded DNA in a sample made up predominantly after duplex structure.