This dissertation focuses on the development of new methods to understand mechanisms of voltage sensing in voltage sensitive proteins. I improved a genetically encoded voltage indicator and developed methods to decrease endogenous fluorescence in Xenopus laevis oocytes. To understand voltage sensor movement, I used a small molecule dye to optically track discrete gating charges and unnatural amino acids to study the importance of charge versus structure of gating charges. Genetically encoded voltage sensors are molecular tools that allow for an optical read out of changes in voltage. I characterized the genetically encoded voltage sensor, ASAP1. I confirmed that is has large fluorescence signal and fast kinetics in response to changes in voltage. However, in the physiological range we found a plateau in the voltage and corresponding fluorescence response. Using a rational design method, I improved ASAP1 and created ASAP-Y. It has an improved response in the physiological range and can follow action potentials in neurons. Next, Xenopus laevis oocytes have variable endogenous fluorescence. However, I developed methods that decreased the endogenous background fluorescence of oocytes, thus improving fluorescence recording conditions. One method relies on injection of a SIK-inhibitor which decreases endogenous fluorescence. The other technique is the injection of synthetic melanin which creates a disc of melanin that decreases endogenous fluorescence. Both of these methods result in better oocyte fluorescence recording conditions. I also investigated the mechanism of voltage sensor movement. To understand the mechanism of voltage sensing, studies have focused on the gating charges. Directly observing the movement of discrete gating charges previously was difficult to achieve. However, through the use of a small positively charged fluorescent dye monobromo(trimethylammonio)bimane (qBBr), we were able to track the first two gating charges of the voltage sensor. Using cut-open voltage clamp fluorimetry, we determined that the voltage sensing domain has a vertical translocation and rotation across the membrane. Moreover, we determined that the path of activation differs from that of deactivation. Further, we have described the physical basis for the Cole-Moore shift. This phenomenon occurs when the initial conditions of the voltage sensor are hyperpolarized before activation, leading to a lag in ion channel opening. This technique, tryptophan-induced quenching, can be applied to understanding other states of the voltage sensor as well as other voltage sensing domains. Finally, we utilized unnatural amino acids (UAA) in several projects. We attempted to observe voltage sensor movement at the level of single molecules. We successfully incorporated Cy3 in the Shaker voltage sensor; however, we never achieved single molecule resolution. We tried to label more internal gating charges with fluorescent unnatural amino acids (fUAA); however, these fUAA never incorporated. We did substitute citrulline for arginines in the voltage sensor to determine the importance of shape versus polarity of an amino acid and find that the loss of charge impacts the fourth gating charge most, whereas gating charges one and three are largely unaffected. Overall, I have employed new methods to understand mechanisms of voltage sensing as well as developed new techniques to improve fluorescence recordings.




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