Articles by Swarna S. Ramaswamy in JoVE
Luminescência Ressonância transferência de energia para estudar mudanças conformacionais na membrana proteínas expressas em células de mamíferos Drew M. Dolino1, Swarna S. Ramaswamy1, Vasanthi Jayaraman1 1Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston Descrevemos aqui um método melhorado de transferência de luminescência Ressonância Energia (lRet) onde é considerado um local de clivagem da protease entre os locais fluoróforo doador e receptor. Esta modificação permite a obtenção de sinais lRet específicas decorrentes de proteínas de membrana de interesse, permitindo o estudo de proteínas de membrana, sem purificação de proteínas.
Other articles by Swarna S. Ramaswamy on PubMed
Proton-mediated Conformational Changes in an Acid-sensing Ion Channel The Journal of Biological Chemistry. Dec, 2013 | Pubmed ID: 24196950 Acid-sensing ion channels are cation channels activated by external protons and play roles in nociception, synaptic transmission, and the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding, there is a conformational change that increases LRET efficiency between the probes at the thumb and finger subdomains in the extracellular domain of acid-sensing ion channels. Additionally, we show that this conformational change is lost upon mutating Asp-238, Glu-239, and Asp-260, which line the finger domains, to alanines. Electrophysiological studies showed that the single mutant D260A shifted the EC50 by 0.2 pH units, the double mutant D238A/E239A shifted the EC50 by 2.5 pH units, and the triple mutant D238A/E239A/D260A exhibited no response to protons despite surface expression. The LRET experiments on D238A/E239A/D260A showed no changes in LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which are involved in carboxyl/carboxylate interactions, are essential for proton-induced conformational changes in the extracellular domain, which in turn are necessary for receptor activation.