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T G Spiro

Publications and source records attributed to T G Spiro.

At least 37 records · Page 2Linked to original sources
PubMed1997

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.

Base Composition↗
PubMed1996

A UV resonance Raman study of hairpin dimer helices of d(A-G)10 at neutral pH containing intercalated dA residues and alternating dG tetrads.

The structure of the oligonucleotide d(A-G)10 in 0.6 M Na+, pH 7.0 has been investigated with UV resonance Raman (UVRR) spectroscopy. Variable wavelength excitation was used to distinguish the spectral contributions of dG and dA residues. Both classes of residues show UVRR hyperchromism with increasing temperature, reflecting unstacking of the bases. The dG residues melt relatively cooperatively with a Tm of approximately 42 degrees C. Unstacking is non-cooperative for the dA residues, increasing linearly between 4 and 80 degrees C. G-tetrads at low temperature are indicated by UVRR frequency shifts of modes associated with C6=O and C2-NH2 of the dG residues, and of vibrations involving N7, all sites of H-bonding. However, there are no indications of interbase H-bonds for the dA residues, showing they do not form H-bonded tetrads. Most of the bases are oriented anti about the glycosyl bond, but at 4 degrees C a fraction of the residues are syn. These results, together with the findings by Shiber et al. [Shiber,M.C., Braswell,E.H., Klump,H. and Fresco,J.R. (1996) Nucleic Acids Res. 24, 5004-5012] that d(A-G)10 under comparable conditions has the molecular weight of a dimer, support a model in which two hairpins interact to form a helical structure with G-tetrads and intercalated dA residues.

Deoxyguanosine↗
PubMed1996

Nanosecond step-scan FTIR spectroscopy of hemoglobin: ligand recombination and protein conformational changes.

Step-scan FTIR spectroscopy with nanosecond time resolution is applied to the photocycle of carbonmonoxy hemoglobin (HbCO). The strong CO stretching band at 1951 cm-1 serves as a convenient monitor of the state of ligation. Both geminate and second-order phases of CO recombination occur at rates which are in excellent agreement with previous visible absorption measurements, showing the molecular mechanisms to be unperturbed by the high protein concentrations (6.7 mM in heme) required for adequate protein signals. While the extent of photolysis (43%) was insufficient to drive the R-->T quaternary transition, the protein TRIR (time-resolved infrared) difference bands (1250-1700 cm-1) nevertheless reveal interesting tertiary dynamics. Most of the bands are fully developed at very early times, possibly preceding the geminate recombination phase (tau = 50 ns). Some bands arise more slowly, however, with a time constant of 0.4 microsecond, reflecting a tertiary motion which is coincident with a quaternary motion previously detected by ultraviolet resonance Raman spectroscopy of fully photolyzed HbCO. Relaxation of the TRIR bands is either faster (tau = approximately 90 microseconds) or slower (tau = approximately 250 microseconds) than CO rebinding (effective time constant of 160 microseconds), suggesting either a distribution of tertiary processes or a chain inequivalence in CO rebinding.

Carbon Monoxide↗
PubMed1995

A UV resonance Raman study of d(A(+)-G)10, a single-stranded helix without stacked or paired bases.

UV resonance Raman spectroscopy has been utilized to directly observe structural features of the recently described nucleic acid single-stranded helix d(A(+)-G)10. An absence of base stacking is confirmed by invariant hypochromic ratios of dominant vibrational modes for the oligomer relative to its constituent monomers as the structure is thermally denatured. The N1 of dA residues is protonated, as determined by similarity to the ring-stretching vibrations for protonated adenine and its derivatives. Selective resonance enhancement of Raman vibrational modes from dA and dG residues shows frequency shifts upon thermal denaturation that confirm the participation of the exocyclic amino of dA but not dG residues in H-bonding. Conformationally sensitive glycosyl bond modes suggest anti residue conformations.

Animals↗
PubMed1995

A methylnickel intermediate in a bimetallic mechanism of acetyl-coenzyme A synthesis by anaerobic bacteria.

Resonance Raman (RR) spectroscopy was used to identify a methylnickel adduct (upsilon Ni-C = 422 wave numbers) of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum. Formed at a nickel/iron-sulfur cluster on CODH called center A, the methylnickel species is the precursor of the methyl group of acetyl-coenzyme A in an anaerobic pathway of carbon monoxide or carbon dioxide fixation. Rapid kinetic and RR studies demonstrated that methylation of nickel occurs by heterolysis of the methyl-cobalt bond (upsilon Co-C = 429 wave numbers) of a methylated corrinoid/iron-sulfur protein. In combination with the earlier finding of an iron-carbonyl adduct at center A, detection of the methylnickel intermediate establishes a bimetallic mechanism for acetyl-coenzyme A synthesis.

Acetyl Coenzyme A↗
PubMed1995

Hemoglobin allostery: resonance Raman spectroscopy of kinetic intermediates.

The end states, R and T, of the allosteric transition in hemoglobin (Hb) are structurally well characterized, but there is little information on intermediate structures along the allosteric pathway. These intermediates were examined by means of time-resolved resonance Raman spectroscopy in the nanosecond-to-microsecond interval after HbCO photolysis. Complementary spectra of the heme group and of the tyrosine and tryptophan residues were recorded during laser excitation at 436 and 230 nanometers. These spectra reveal a sequence of interleaved tertiary and quaternary motions during the photocycle, motions involving the proximal and distal helices, and the alpha 1 beta 2 subunit interface. This sequence leads to a modified form of the T state, in which the alpha 1 beta 2 interface is deformed as a result of two carbon monoxide molecules binding to the same dimer within the tetramer.

Allosteric Regulation↗
PubMed1995

Stereoelectronic activation of methylenetetrahydrofolate by thymidylate synthase: resonance Raman spectroscopic evidence.

Resonance Raman (RR) spectra are reported for the ternary complex of Escherichia coli thymidylate synthase with the cofactor 5,10-methylenetetrahydrofolate (CH2-H4-folate) and the inhibitor 5-fluoro-2'-deoxyuridylate, excited at 337 or 356 nm, in resonance with perturbed absorption bands of the p-aminobenzoylglutamate (PABA-Glu) portion of the cofactor. For comparison, RR spectra were obtained with 260 nm excitation for PABA-Glu in various solvents, and for CH2H4-folate and H4-folate in aqueous solution. These reference spectra are assigned to modes of PABA-Glu in its benzenoid form. The ternary complex RR spectra are very different, however, and are assigned, with the aid of isotopic data, to the PABA-Glu in a predominantly quinoid form. Similar spectra were obtained for the ternary complexes of the E58Q and K48Q mutants, indicating that neither Glu58 nor Lys48 are essential for maintaining the quinoid structure, even though their side chains complement the dipolar charge distribution of the quinoid form of PABA-Glu. Since these are the only charged residues in the PABA-Glu vicinity, electrostatic stabilization is not essential to maintenance of the quinoid structure. It is proposed that quinoid formation results from steric forces, probably resulting from the protein conformation change known to accompany cofactor binding, which enforce coplanarity of the PABA-Glu ring and substituents. This stereoelectronic change activates the cofactor by opening the methylene bridge. A second RR spectrum of the ternary complex, previously proposed to reflect an alternate structure, is shown to result instead from irreversible formation of a laser-induced photoproduct.

Escherichia coli↗
PubMed1995

Resonance Raman characterization of the binary and ternary complexes of thymidylate synthase with 5-nitrodeoxyuridylate.

Resonance Raman (RR) spectra are reported for the binary complex of Escherichia coli thymidylate synthase (TS) with the substrate analog inhibitor 5-nitrodeoxyuridylate (NDU). The TS/NDU binary complex RR spectrum shows many similarities to the RR spectra of thiol adducts of NDU or of 5-nitro-1-methyluracil formed in solution, providing strong evidence in support of the formation of a covalent link between Cys146 of TS and C6 of NDU. Spectral differences between the model compounds and the binary complex reflect the consequences of fixing the conformations of the uracil and ribose rings at the enzyme active site. The RR spectra of the ternary complexes of TS/NDU with either tetrahydrofolate (H4-folate) or the cofactor 5,10-methylenetetrahydrofolate (CH2H4-folate) show that a covalent link is not formed between C11 of CH2H4-folate and C5 of NDU. Neither does the methylene bridge of CH2H4-folate remain intact in the ternary complex; either CH2H4-folate is present as the N5 iminium cation species or the methylene group is lost as formaldehyde. A shift in the NO2 symmetric stretching frequency in the ternary complex indicates expulsion of water molecules from the region of the NO2 group by the cofactor.

Deoxyuracil Nucleotides↗
PubMed1995

FT-IR difference spectroscopy of hemoglobins A and Kempsey: evidence that a key quaternary interaction induces protonation of Asp beta 99.

Fourier transform infrared difference spectra are reported for the CO adduct of human hemoglobin versus deoxyHb, in H2O and D2O. In addition to the well-known CO stretching and S-H(D) stretching bands, the difference spectra reveal numerous bands in the 1200-1700 cm-1 region, a number of which are assigned. Several amide modes are identified via their frequencies and D2O sensitivities. Bands arising from histidine protonation have also been found via comparison of the difference spectra at different pH(D) values, with the aid of aqueous histidine spectra. Of particular interest is the observation of a negative band at 1697 cm-1, which is assigned to the C = O stretch of carboxylic acid. This carboxylic acid is tentatively identified as the side chain of Asp beta 99, because it is missing in the difference spectrum of Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn. Asp beta 99 forms a critical contact with Tyr alpha 42 across the alpha 1 beta 2 interface in deoxyHb, which is broken upon ligation. Protonation of Asp beta 99 in deoxyHb is consistent with UV resonance Raman evidence that Tyr alpha 42 is the acceptor rather than the donor of the quaternary H-bond.

Adult↗
PubMed1995

Structure of a third cooperativity state of hemoglobin: ultraviolet resonance Raman spectroscopy of cyanomethemoglobin ligation microstates.

Ultraviolet resonance Raman (UVRR) spectra have been obtained for cyanometHb (Hb = hemoglobin) hybrid tetramers representing several ligation microstates, using dimer interchange techniques and spectral subtraction. Relative to fully ligated tetramers, Hb(CN)4, the monoligated hybrids and deoxyHb all show fully developed T/R difference UVRR bands which are associated with T state quaternary contacts across the alpha 1 beta 2 interface, involving the Trp beta 37 and Tyr alpha 42 residues. Triligated species show quite different signals, arising from the interior residues Trp alpha 14 and/or beta 15. From earlier studies, these Rdeoxy signals are attributed to E helix displacement toward the heme in deoxy subunits within R state tetramers, resulting in weakened Trp H-bonds. Asymmetric diligated hybrids, containing both ligands in the same dimer unit, show signals characteristic of the T quaternary contacts, but they are attenuated by 40%. An equilibrium mixture of T and R state molecules is ruled out by the absence of significantly strong Rdeoxy difference bands. Rather, the spectral attenuation is attributed to weakening of the T state contacts at the alpha 1 beta 2 interface. This interpretation is supported by previous observations that the mutational pattern of free energy perturbations for the asymmetric hybrid is T-like and not that of a T/R equilibrium or an R-like state. The asymmetric hybrid represents a third cooperativity state, T', having a T quaternary arrangement of the subunits but a deformed alpha 1 beta 2 interface, with weakened contacts.

Hemoglobins↗
PubMed1995

Ultraviolet resonance Raman spectroscopy of delta 5-3-ketosteroid isomerase revisited: substrate polarization by active-site residues.

The delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes extremely rapid conversion of delta 5- to delta 4-3-ketosteroids by a conservative intramolecular proton transfer via an enolic intermediate. The competitive inhibitor 19-nortestosterone displays marked spectroscopic changes upon binding to the enzyme, but the mechanisms responsible for these changes have not been unequivocally established. Ultraviolet resonance Raman (UVRR) spectra are reported for 19-nortestosterone in acid solutions and for this ligand when bound to delta 5-3-ketosteroid isomerase, as well as to its D38N and Y14F/D38N mutants. The frequencies of UVRR bands associated with C = O and C = C stretching can be used to monitor the state of polarization of the enone fragment of the steroid and the effects of the catalytic side chains, Tyr-14 and Asp-38, on these polarizations. Strong polarization is indicated by marked frequency downshifts of the C = O and C = C bands in the native protein; the downshifts are diminished by the mutations of these catalytic residues. The lower polarizing effects of the Y14F and D38N single mutants and the Y14F/D38N double mutant indicate that most of the polarization of the conjugated ketone is attributable to hydrogen-bond donation by the hydroxyl group of Tyr-14. A smaller contribution of Asp-38 is detected which is, in part, cooperative with that of Tyr-14. Reference spectra of hydrogen-bonded and protonated forms of 19-nortestosterone are reassigned, on the basis of the species identification of D. C. Hawkinson and R. M. Pollack [(1993) Biochemistry 32, 694-698].(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Resonance Raman spectroscopic characterization of the molybdopterin active site of DMSO reductase.

Resonance Raman spectra are compared for Rhodobacter sphaeroides dimethyl sulfoxide reductase, an enzyme containing a molybdopterin cofactor, and two model compounds, I and II, which have pterin and quinoxaline, respectively, attached to a Cp2Mo[IV]-dithiolene chelate [Cp = cyclopentadienyl]. The effect of 34S incorporation was also determined. Several bands in the 200-500 cm-1 region show remarkably similar patterns of frequencies and isotope shifts between protein and models: a band at 351 cm-1 shifts 6-8 cm-1, and bands at lower and higher frequencies show smaller shifts upon 34S substitution. A normal coordinate analysis on II indicates the 351 cm-1 mode to be the symmetric Mo-S[dithiolene] stretch and the remaining low-frequency modes to contain contributions from deformations of the quinoxaline ring as well as from Mo-S stretching. The similarity in the low-frequency spectra between the model compounds and the enzyme strongly supports a dithiolene chelate as the mode of Mo-pterin interaction in the cofactor. Resonance enhancement of both high- and low-frequency quinoxaline or pterin modes is observed for both model compounds, implicating the heterocyclic rings as part of the electronic system involved in the Mo-dithiolene charge transfer transitions. RR spectra of 6-methylpterin and biopterin are reported and used to identify the pterin and quinoxaline high-frequency bands in the model compound spectra. The dithiolene C = C stretch is tentatively assigned to bands at 1506 cm-1 in I and 1515 cm-1 in II.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Secondary and tertiary structure of the A-state of cytochrome c from resonance Raman spectroscopy.

Ferricytochrome c can be converted to the partially folded A-state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A-state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease in amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230-nm spectra contain information on vibrational modes of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chain remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a show disruption of tertiary hydrogen bonding for the Tyr 48, 67, and 74 side chains. The high-wavenumber region of the 406.7-nm resonance Raman spectrum reveals a mixed spin heme iron atom, which arises from axial coordination to His 18 and a water molecule. The low-frequency spectral region reports on heme distortions and indicates a reduced degree of interaction between the heme and the polypeptide chain. A structural model for the A-state is proposed in which a folded protein subdomain, consisting of the heme and the N-terminal, C-terminal, and 60s helices, is stabilized through nonbonding interactions between helices and with the heme.

Animals↗
PubMed1994

Modeling the hemoglobin switchpoint with cyanomet valency hybrids: Raman spectroscopic probes of tertiary and quaternary structure.

Hybrid hemoglobins with cyanomet hemes in the alpha or in the beta chains have been investigated by resonance Raman (RR) spectroscopy, using ultraviolet (230 nm) and visible (441.6 nm) excitation. For the CO adducts, the UVRR spectra are identical with that of native HbCO, showing the tyrosine and tryptophan signals to be insensitive to ligand substitution within the R state. In the absence of CO, the doubly ligated hybrids show differences in the UVRR spectra, relative to the CO adducts, which are the superposition of two difference spectra: (1) the T-R difference spectrum obtained by subtracting the spectrum of HbCO from that of deoxyHb and (2) a perturbed R state spectrum, characteristic of deligated chains within the R state. These spectra arise from alterations, respectively, in the quaternary contacts of interface aromatic residues and in the tertiary contacts of interior aromatic residues. From the amplitudes of the difference spectra, the T state population was determined to be 30% for (alpha FeII beta FeIIICN)2 and 43% for (alpha FeIIICN beta FeII)2, in good agreement with the kinetic analysis of Cassoly and Gibson [Cassoly, R., & Gibson, Q. H. (1972) J. Biol. Chem. 247, 7332]. Addition of inositol hexaphosphate (IHP) increased the T state population, but only by a modest amount, to 40 and 53%, respectively, in contrast to the frequent assumption that the T state conversion is quantitative in the presence of IHP. Since current understanding of the quaternary state dependence of the Fe-histidine stretching frequency is based on that assumption, the RR band envelope for this vibration was reexamined.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobins↗
PubMed1994

Nanosecond dynamics of the R-->T transition in hemoglobin: ultraviolet Raman studies.

Pulse-probe transient Raman spectroscopy, with probe excitation at 230 nanometers, reveals changes in signals arising from tyrosine and tryptophan residues of the hemoglobin molecule as it moves from the relaxed (R) to the tense (T) state after photodeligation. Signals associated with intersubunit contacts in the T state develop in about 10 microseconds but are preceded by quite different signals, which reach maximum amplitude in about 50 nanoseconds. These signals involve the interior tryptophan residues that bridge the A and E helices by means of H bonds between the indole rings and serine or threonine side chains. Alterations of the H bond strengths, as a result of interhelix motions, can account for the signals. A model is proposed here in which loss of the ligand from the heme binding pocket is concerted with inward motion of the adjacent E helix; this motion, along with a complementary motion of the proximal F helix, transmits the energy associated with heme deligation to the subunit interfaces, leading to the T state rearrangement.

Carboxyhemoglobin↗