RESUMO
Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r=0.65-0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations.
Assuntos
Modelos Químicos , Modelos Moleculares , Modelos Estatísticos , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/ultraestrutura , Simulação por Computador , Estabilidade Enzimática , Método de Monte Carlo , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/genética , Temperatura de TransiçãoRESUMO
The tetramolecular PNA quadruplex motif has been probed using a dynamic covalent chemistry (DCC) approach to create and characterize a bimolecular PNA quadruplex.
Assuntos
DNA/síntese química , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Quadruplex G , MétodosAssuntos
DNA/química , Guanina/química , Moldes Genéticos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Técnicas de Química Combinatória , DNA/metabolismo , Quadruplex G , Glutationa/química , Cinética , Ligantes , Conformação de Ácido Nucleico , Oxirredução , Ressonância de Plasmônio de SuperfícieRESUMO
Here we report the synthesis of a novel PNA based neocuproine.Zn RNA cleaving agent; we demonstrate that such agents sequence specifically cleave a synthetic RNA target and in particular the RNA component of human telomerase.
Assuntos
Ácidos Nucleicos Peptídicos/química , Fenantrolinas/química , RNA não Traduzido/química , Telomerase/química , Zinco/química , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Humanos , Ácidos Nucleicos Peptídicos/farmacologia , Fenantrolinas/farmacologia , RNA , RNA Longo não Codificante , RNA não Traduzido/metabolismo , Telomerase/metabolismoRESUMO
Use of anabolic-androgenic steroids (AASs) is becoming increasingly popular among adolescent girls, yet the effects of AASs on female physiology and development are not well understood. The present study compared the effects of chronic exposure to three individual AASs, stanozolol (0.05-5 mg/kg), 17alpha-methyltestosterone (0.5-5 mg/kg), and methandrostenolone (0.5-5 mg/kg) on the onset of puberty and estrous cyclicity in the rat. Female rats received daily injections of AASs for 30 days (Postnatal Day [PN] 21-51). Rats receiving the highest dose of each of the AASs (5 mg/kg) displayed vaginal opening at a younger age than rats receiving the oil vehicle. The day of first vaginal estrus was delayed in rats receiving stanozolol (5 mg/kg) or 17alpha-methyltestosterone (0.5-5 mg/kg) but not in rats receiving methandrostenolone. At the highest dose (5 mg/kg), each of the AASs reduced the incidence of regular estrous cyclicity during the treatment period. Concurrent administration (on PN21-51) of the androgen receptor antagonist, flutamide (10 mg/kg, twice daily), reversed the effects of 17alpha-methyltestosterone (5 mg/kg) on vaginal opening. Flutamide administration also eliminated the effects of stanozolol (5 mg/kg) and 17alpha-methyltestosterone (5 mg/kg) on the day of first vaginal estrus. In contrast, rats receiving flutamide and methandrostenolone (5 mg/kg) exhibited first vaginal estrus earlier than controls. The present results indicate that chronic exposure to AASs during development has deleterious effects on the female neuroendocrine axis and that these effects appear be mediated via multiple mechanisms.