FTIR characterization of heterocycles lumazine and violapterin in solution: effects of solvent on anionic forms.

Fourier transform infrared (FTIR) spectra have been obtained from solution samples of the heterocycles uracil, lumazine, and violapterin and reveal interpretable carbonyl stretching frequencies. Spectra of conjugate bases of lumazine and violapterin demonstrate decreases in these carbonyl stretching frequencies upon ionization. Based on isotopic shifts from amide deuterated analogs, semiempirical QCFF/PI calculations were used to assign the vibrational frequencies in the region 1100-1800 cm-1 observed from samples in dimethylsulfoxide (DMSO) and aqueous solutions to specific normal modes. The observed deuterium shifts and the calculations suggest that, in some cases, N-H bending motions are coupled to the C=O stretching motions of the pyrimidine ring. These data suggest that for lumazine anions a change in solvent can significantly change the mixing of the N-H bending and C=O stretching vibrational motions. This implies that vibrational analysis for lumazine species in relatively noninteracting media like nonpolar solvents, mulls or pellets cannot necessarily be transferred to the system when it is dissolved in a polar, hydrogen-bonding solvent such as water. Although other explanations can be offered, our vibrational analysis suggests that the changes in normal mode composition of the predominantly C=O stretching vibrations of lumazine anion on going from dimethylsulfoxide to water solution are consistent with a change in the predominant tautomer of the heterocycle. This change appears to correspond to a shifting of the location of the remaining acidic proton to a different ring nitrogen atom. This interpretation is of interest in view of recent ab initio calculations which suggest that proton shifts may occur during the hydroxylation of lumazine as mediated by the enzyme xanthine oxidase.

Stereospecific reaction of muscle fiber proteins with the 5' or 6' isomer of (iodoacetamido)tetramethylrhodamine.

The labeling of muscle fiber proteins with iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (S1) but with different rates, and only the 5'-IATR alters K(+)-EDTA- and Ca(2+)-activated ATPases. Absorption spectroscopic and ATPase studies of probe stoichiometry indicate that for 5'-IATR there are two probes per myosin sulfhydryl 1 (SH1). Quantitative fluorograms of the SDS-PAGE gels confirm that there are one covalent and one noncovalent probe per SH1 when S1 is labeled with 5'-IATR (5'-IATR-S1) and that there are one covalent and two noncovalent probes per S1 when S1 is labeled with 6'-IATR (6'-IATR-S1). The 5'- and 6'-IATR probes have similar fluorescent lifetimes when bound to S1, but quenching studies with potassium iodide show that 5'-IATR-S1 has a single class of strongly bound chromophores while 6'-IATR-S1 has two or more classes of chromophores. It is possible that 5'-IATR labels SH1 as a dimer. The polarization anisotropies of 5'- and 6'-IATR-S1 indicate that 5'-IATR is immobilized, while 6'-IATR is moving independently, on the surface of S1. The emission spectrum from 5'-IATR-S1 is unaffected by the addition of MgATP, while 6'-IATR-S1 shows a spectral shift and total intensity change. When labeling muscle fibers, 5'-IATR labels myosin SH1 and differentiates between the fiber physiological states by indicating cross-bridge rotation in quantitative agreement with previous results [Burghardt et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515]. 6'-IATR reacts preferentially with actin in muscle fibers and does not differentiate between fiber physiological states as expected for an actin probe. The stereospecificity of the rhodamine isomers for SH1 indicates features of the local protein structure. The experimental results are used with theoretical methods for determining molecular structure to suggest a qualitative scheme for the specific interaction of 5'-IATR with its binding pocket on the surface of S1.

Protein: nucleic acid interactions. I. Electronic structures of cytosine, indole, and guanine complexes.

Low singlet transition energies and line strengths were calculated for the cytosine:indole:guanine complex by the INDO/1S-CI method. The chromophores were arranged in three sets of 270 intercalating geometries. Calculations were executed in the supermolecule model with single excited configurations. Errors due to basis set extension and incomplete configuration representation were assessed, for all chromophore pairs, by full BSSE correction calculations and inclusion of double-excited configurations. The intercalation-induced perturbations of the principal transitions are characterized by but not limited to (a) a decrease in strength of [pi*,pi] transitions, (b) increase in strength in [pi*,n] transitions, (c) splitting of [pi*,pi] transitions into components of unequal strength, and (d) energy and strength dependence in mixed transitions on rise and shift movements of the nucleic acid bases. These predictions are in accord with absorption, fluorescence emission, and scattering, and resonance Raman spectroscopic data on oligonucleotides and analogous aromatic complexes. The calculations suggest that major differences in intercalating coordinations are discernible in the near-uv spectroscopic domain of proteins and nucleic acids.

Electronic states of the indole-acrylamide molecular pair.

A model is suggested for tryptophan fluorescence quenching by acrylamide based on the prediction that acrylamide can absorb a photon from the excited indole moiety and then dissipate the optical energy into a sink of fast exchanging conformations. Semiempirical electronic structure calculations of the indole-acrylamide pair indicate little actual intermolecular orbital mixing at van der Waals contact distances. However, the two lowest singlet transitions of the molecular pair, assigned to the acrylamide (pi *)----n(O) line and to the indole 1Lb----1A1 line, respectively, vary significantly in energies and in transition and excited state moments with the geometry of interaction between the two entities. The distribution of optimal quenching coordinations depends separately on the benzene and pyrrole portions and has a distinctly non-spherical shape at these distances.

Electronic transitions in molecules in static external fields. I. Indole and Trp-59 in ribonuclease T1.

Electronic transition properties of indole perturbed by its environment were calculated by use of quantum-mechanical semi-empirical numerical methods. The environment was represented by a discrete set of charges placed at different positions around the indole ring. Wavelength shifts and transition intensity changes in indole were evaluated for several, specifically modeled geometries of external charges. This methodology was employed to estimate the extent of spectroscopic changes induced by small nonprotein polar species on the Trp-59 residue in the anisotropic environment of the protein ribonuclease T1. The geometry of the residue environment was obtained from dynamically equilibrated X-ray crystallographic data of the protein.