Presentation Type

Poster

Abstract

G-proteins are important in regulating several cellular processes, which when defective, have been associated with several endocrinal disorders. G-protein associated disorders arise from either a disability to initiate proper downstream signaling, a deficiency in the ability to terminate the signal, or from a reduced level of G-proteins present in the cell. Resistance to inhibitors of cholinesterase 8 (Ric-8A) is an ∼60-kDa cytosolic protein that functions as a molecular chaperone for heterotrimeric G-protein α subunits in vivo, and functions as a GEF (Guanine Nucleotide Exchange Factor) in vitro. The nucleotide exchange activity of Ric-8A is poorly understood. In this study, we aim to measure the rates of conformational fluctuation undergone by Gαi1 during Ric-8A catalyzed nucleotide exchange by following signal changes in FRET (Förster Resonance Energy Transfer) using rapid mixing stopped-flow fluorescence spectroscopy. As fluorescence is distance dependent, changes in fluorescence between FRET pairs is indicative of protein conformational changes. Several constructs of Hexa I-Gαi, containing two cysteine mutations at various locations in the Ras or Helical domain, are labeled with Alexa dye pairs in this experiment. The stopped-flow enables rapid addition of Ric-8A, allowing the FRET signal to be monitored upon initiation of nucleotide exchange. A decrease in FRET is expected upon addition of Ric-8A, as a conformational change occurs increasing the distance between the two labeled domains. Understanding the conformational effects that Ric-8A has on the Gα subunit, can lead to future therapeutic treatment of G-protein associated diseases.

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Life Sciences

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Apr 28th, 3:00 PM Apr 28th, 4:00 PM

Conformational Changes of Gai1 nucleotide exchange catalyzed by Ric-8A

UC South Ballroom

G-proteins are important in regulating several cellular processes, which when defective, have been associated with several endocrinal disorders. G-protein associated disorders arise from either a disability to initiate proper downstream signaling, a deficiency in the ability to terminate the signal, or from a reduced level of G-proteins present in the cell. Resistance to inhibitors of cholinesterase 8 (Ric-8A) is an ∼60-kDa cytosolic protein that functions as a molecular chaperone for heterotrimeric G-protein α subunits in vivo, and functions as a GEF (Guanine Nucleotide Exchange Factor) in vitro. The nucleotide exchange activity of Ric-8A is poorly understood. In this study, we aim to measure the rates of conformational fluctuation undergone by Gαi1 during Ric-8A catalyzed nucleotide exchange by following signal changes in FRET (Förster Resonance Energy Transfer) using rapid mixing stopped-flow fluorescence spectroscopy. As fluorescence is distance dependent, changes in fluorescence between FRET pairs is indicative of protein conformational changes. Several constructs of Hexa I-Gαi, containing two cysteine mutations at various locations in the Ras or Helical domain, are labeled with Alexa dye pairs in this experiment. The stopped-flow enables rapid addition of Ric-8A, allowing the FRET signal to be monitored upon initiation of nucleotide exchange. A decrease in FRET is expected upon addition of Ric-8A, as a conformational change occurs increasing the distance between the two labeled domains. Understanding the conformational effects that Ric-8A has on the Gα subunit, can lead to future therapeutic treatment of G-protein associated diseases.