RESUMO
Fourier transform infrared (FTIR) spectroscopy and a library of FTIR marker bands have been used to examine the structure and relative flexibilities conferred by different flanking sequences on the EcoRI binding site. This approach allowed us to examine unique peaks and subtle changes in the spectra of d(AAAGAATTCTTT)(2), d(TTCGAATTCGAA)(2), and d(CGCGAATTCGCG)(2) and thereby identify local changes in base pairing, base stacking, backbone conformation, glycosidic bond rotation, and sugar puckering in the studied sequences. The changes in flanking sequences induce differences in the sugar puckers, glycosidic bond rotation, and backbone conformations. Varying levels of local flexibility are observed within the sequences in agreement with previous biological activity assays. The results also provide supporting evidence for the presence of a splay in the G(4)-C(9) base pair of the EcoRI binding site and a potential pocket of flexibility at the G(4) cleavage site that have been proposed in the literature. In sum, we have demonstrated that FTIR is a powerful methodology for studying the effect of flanking sequences on DNA structure and flexibility, for it can provide information about the local structure of the nucleic acid and the overall relative flexibilities conferred by different flanking sequences.
Assuntos
DNA/química , Sequência de Bases , Sítios de Ligação , Desoxirribose/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fosfatos/química , Maleabilidade , Espectroscopia de Infravermelho com Transformada de Fourier , VibraçãoRESUMO
The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein--DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here, we use 31P dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2H NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps, but this effect is localized to only near neighbors and base-pairing partners. Last, we have been able to characterize the percent BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the noncanonical BII conformation, even at low temperatures. Our results demonstrate that 31P dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events.
Assuntos
DNA/química , Espectroscopia de Ressonância Magnética/métodos , Radioisótopos de Fósforo/uso terapêutico , Algoritmos , Sítios de Ligação , Biofísica/métodos , Modelos Teóricos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Proteínas/química , Temperatura , TermodinâmicaRESUMO
We report use of dynamic nuclear magnetic resonance (NMR) to measure the amide rotational barrier in isonicotinamide. A significant challenge to obtaining good transition rates from dynamic NMR data is suppression of errors due to inherent line widths associated with transverse relaxation. We address this challenge with a fitting procedure that incorporates transverse relaxation over the temperature range of interest simply and reliably. The fitting model is nonlinear in only one of the fit parameters, namely, the activation enthalpy. This reduces parameter estimation to solution of a single transcendental equation, which avoids both a fine search over a multidimensional parameter space and extrapolation of a "limiting line width" solely from slow-exchange data. The activation enthalpy Delta H++ measured for isonicotinamide, +14.1 +/- 0.2 kcal/mol, falls between those of its regioisomers picolinamide and nicotinamide, which were reported in an earlier study. In that study, ab initio calculations of the rotational barriers helped to discern the relative importance of steric, electronic, and hydrogen-bonding effects in this biochemically significant combination of pyridine-ring and carboxamide moieties. A direct comparison between isonicotinamide and nicotinamide, where steric and hydrogen-bonding effects differ only slightly, permits a closer study of electronic considerations.
Assuntos
Amidas/química , Niacinamida/química , Algoritmos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Ácidos Picolínicos/química , Teoria Quântica , RotaçãoRESUMO
By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of [zeta-(15)N]lys-, [12-(13)C]retinal-, and [14,15-(13)C]retinal-labeled D85N samples indicate a deprotonated, 13-cis,15-anti chromophore. On the other hand, at neutral pH, the NMR spectra of D85N show a mixture of protonated Schiff base species similar to that seen in the wild-type protein at low pH, and more complex than the two-state mixture of 13-cis,15-syn, and all-trans isomers found in the dark-adapted wild-type protein. These results lead to several conclusions. First, the reversible titration of order in the D85N chromophore indicates that electrostatic interactions have a major influence on events in the active site. More specifically, whereas a straight chromophore is preferred when the Schiff base and residue 85 are oppositely charged, a bent chromophore is found when both the Schiff base and residue 85 are electrically neutral, even in the dark. Thus a "bent" binding pocket is formed without photoisomerization of the chromophore. On the other hand, when photoisomerization from the straight all-trans,15-anti configuration to the bent 13-cis,15-anti does occur, reciprocal thermodynamic linkage dictates that neutralization of the SB and D85 (by proton transfer from the former to the latter) will result. Second, the similarity between the chromophore chemical shifts in D85N at alkaline pH and those found previously in the M(n) intermediate of the wild-type protein indicate that the latter has a thoroughly relaxed chromophore like the subsequent N intermediate. By comparison, indications of L-like distortion are found for the chromophore of the M(o) state. Thus, chromophore strain is released in the M(o)-->M(n) transition, probably coincident with, and perhaps instrumental to, the change in the connectivity of the Schiff base from the extracellular side of the membrane to the cytoplasmic side. Because the nitrogen chemical shifts of the Schiff base indicate interaction with a hydrogen-bond donor in both M states, it is possible that a water molecule travels with the Schiff base as it switches connectivity. If so, the protein is acting as an inward-driven hydroxyl pump (analogous to halorhodopsin) rather than an outward-driven proton pump. Third, the presence of a significant C [double bond] N syn component in D85N at neutral pH suggests that rapid deprotonation of D85 is necessary at the end of the wild-type photocycle to avoid the generation of nonfunctional C [double bond] N syn species.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Mutação , Sítios de Ligação , Carbono/química , Citoplasma/metabolismo , Concentração de Íons de Hidrogênio , Luz , Lisina/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Prótons , Retinoides/química , TemperaturaRESUMO
Pyridine carboxamides are a class of medicinal agents with activity that includes the reduction of iron-induced renal damage, the regulation of nicotinamidase activity, and radio- and chemosensitization. Such pharmacological activities, and the prevalence of the carboxamide moiety and the importance of amide rotations in biology, motivate detailed investigation of energetics in these systems. In this study, we report the use of dynamic nuclear magnetic resonance to measure the amide rotational barriers in the pyridine carboxamides picolinamide and nicotinamide. The activation enthalpies and entropies of DeltaH++ = 12.9 +/- 0.3 kcal/mol and DeltaS++ = -7.7 +/- 0.9 cal/mol K for nicotinamide and DeltaH++ = 18.3 +/- 0.4 kcal/mol and DeltaS++ = +1.3 +/- 1.0 cal/mol K for picolinamide report a substantial energetic difference for these regioisomers. Ab initio calculations of the rotational barriers are in good agreement with the experimentally determined values and help partition the 5.4 kcal/mol enthalpy difference into its major contributions. Of principal importance are the variations in steric interactions in the ground states of picolinamide and nicotinamide, superior pi electron donation from the pyridine ring in the transition state of nicotinamide, and an intramolecular hydrogen bond in the ground state of picolinamide.