RESUMO
The influence of pressure on the equilibrium between five-(5c) and six-coordination (6c) forms in neuroglobin (Ngb) and myoglobin (Mb) has been examined by means of molecular dynamics (MD) simulations at normal and high pressure. The results show that the main effect of high pressure is to reduce the protein mobility without altering the structure in a significant manner. Moreover, our data suggest that the equilibrium between 5c and 6c states in globins is largely controlled by the structure and dynamics of the C-D region. Finally, in agreement with the available experimental data, the free energy profiles obtained from steered MD for both proteins indicate that high pressure enhances hexacoordination. In Ngb, the shift in equilibrium is mainly related to an increase in the 6c-->5c transition barrier, whereas in Mb such a shift is primarily due to a destabilization of the 5c state.
Assuntos
Globinas/química , Mioglobina/química , Proteínas do Tecido Nervoso/química , Animais , Simulação por Computador , Interpretação Estatística de Dados , Entropia , Humanos , Camundongos , Modelos Moleculares , Neuroglobina , Pressão , Conformação Proteica , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
The C-terminal DNA binding domain of the E2 protein is involved in transcriptional regulation and DNA replication in papillomaviruses. At low ionic strength, the domain has a tendency to form aggregates, a process readily reversible by the addition of salt. While fluorescence anisotropy measurements show a 1:1 stoichiometry at pH 5.5, we observed that a second HPV-16 E2 C-terminal dimer can bind per DNA site at pH 7.0. This was confirmed by displacement of bis-ANS binding, tryptophan fluorescence, native electrophoresis, and circular dichroism. The two binding events are nonequivalent, with a high-affinity binding involving one E2C dimer per DNA molecule with a K(D) of 0.18 +/- 0.02 nM and a lower affinity binding mode of 2.0 +/- 0.2 nM. The bovine (BPV-1) E2 C-terminal domain binds to an HPV-16 E2 site with 350-fold lower affinity than the human cognate domain and binds 7-fold less tightly even to a bovine-derived DNA site. The ability to discriminate between cognate and noncognate sequences is 50-fold higher for the human domain, and the latter is 180-fold better than the bovine at discriminating specific from nonspecific DNA. A substantial conformational change in bound DNA is observed by near-UV circular dichroism. The bovine domain imposes a different DNA conformation than that caused by the human counterpart, which could be explained by a more pronounced bent. Structure-function differences and biochemical properties of the complexes depend on the protein domain rather than on the DNA, in line with crystallographic evidence. Despite the strong sequence homology and overall folding topology, the differences observed may explain the distinctive transcriptional regulation in bovine and human viruses.
Assuntos
Proteínas E2 de Adenovirus/química , Papillomavirus Bovino 1/química , Sequência Consenso , DNA/química , Conformação de Ácido Nucleico , Papillomaviridae/química , Fragmentos de Peptídeos/química , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/metabolismo , Animais , Sequência de Bases , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/metabolismo , Bovinos , Dicroísmo Circular , DNA/metabolismo , Humanos , Concentração Osmolar , Papillomaviridae/genética , Papillomaviridae/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Dobramento de Proteína , Estrutura Terciária de Proteína/genética , SoluçõesRESUMO
OBJECTIVE: This study had two objectives: (1) to assess infant behavior by using the NICU Network Neurobehavioral Scale (NNNS), an assessment designed specifically for prenatally drug-exposed infants; and (2) to control for the effects of polydrug use involving alcohol, marijuana, and cigarettes on the neurobehavioral status of the newborn infant. METHODS: The subjects and controls in this study were full-term infants of appropriate gestational age with no medical problems. At 1 to 2 days of age, 20 infants exposed to cocaine, alcohol, marijuana, and cigarettes; 17 infants exposed to alcohol and/or marijuana and cigarettes; and 20 drug-free infants were evaluated by using the Neonatal Intensive Care Unit Network Neurobehavioral Scale. The data were reduced to reflect clinically defined categories of neurobehavioral function and were analyzed by using analysis of variance and chi 2 statistics. RESULTS: Cocaine-exposed infants showed increased tone and motor activity, more jerky movements, startles, tremors, back arching, and signs of central nervous system and visual stress than unexposed infants. They also showed poorer visual and auditory following. There were no differences in how the examination was administered to cocaine-exposed and nonexposed infants. Reduced birth weight and length were also observed in cocaine-exposed infants. CONCLUSION: Differences attributable to cocaine-exposed infants were related to muscle tone and motor performance, following during orientation, and signs of stress. However, cocaine-exposed infants were not more difficult to test, nor did they require an alteration in the examination. Both neurobehavioral patterns of excitability and lethargy were observed. Findings may have been due to the synergistic effects of cocaine with alcohol and marijuana.
