Structural dynamics in the resting and activated states of the voltage sensor of Ci-VSP from dipolar distance measurements
Carlos A. Villalba-Galea: 0000-0002-6489-4651
Conference Title/Conference Publication
Biophysical Society 56th Annual Meeting
San Diego, CA
February 25-29, 2012
Date of Presentation
3, Supplement 1
The mechanism of electromechanical transduction in voltage sensing domains remains controversial. Here, we have probed the conformation of the voltage sensor of Ci-VSP in different functional states by means of EPR-based distance measurements. Ci-VSP is a voltage-sensing phosphatase from Ciona intestinalis. Although it is coupled to a cytoplasmic phosphatase, its voltage-sensing domain (VSD) is homologous to voltage sensors found in voltage-gated ion channels. It therefore serves as an excellent model to study voltage sensor movement independent of the interaction with pore domain. On the basis of voltage dependence of Ci-VSP sensing currents (Q-V curves), it is agreed that, at 0 mV, the S4 of wild-type Ci-VSP is in the resting conformation (down state). The arginine at position 217, located in the extracellular end of S4, has a strong effect on the voltage dependence of Ci-VSP sensing currents. Mutations at arginine 217 with a neutral or negative residue (R217Q or R217E), lead to a large leftward shifts in the Q-V curve so that, at 0 mV, the sensor is in the activated conformation (up state). This provides a unique opportunity to monitor the conformational differences in the VSD between resting and activated states in the absence of membrane potential. We expressed and purified a series of double cysteine mutants in the isolated voltage sensor (S1 to S4) of Ci-VSP in wild-type and R217E backgrounds, and measured distances using CW-based dipolar broadenings (for short distances, 8 to 20 Å) and double electron-electron resonance (DEER) spectroscopy (for longer distances, 20 to 50 Å). Our preliminary analysis of the distance measurements suggest defined conformational differences between resting and activated states of the VSD.
Raghuraman, H.; Wanderling, Sherry; Villalba-Galea, Carlos A.; Perozo, Eduardo; and Li, Qufei, "Structural dynamics in the resting and activated states of the voltage sensor of Ci-VSP from dipolar distance measurements" (2012). School of Pharmacy Faculty Presentations. 425.