| 2018 — 2019 |
Iwanicki, Martin Joshua |
F31Activity Code Description:
To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Engineering Novel Neuronal Optical Voltage-Sensing Probes Using Synthetic Proteins @ University of Pennsylvania
Understanding the structure and dynamics of the neural circuitry that dictates complex behavior is one of the long-established goals of neuroscience. The ability to visualize and comprehend how interneuronal interactions generate network dynamics in processes such as learning, memory, sensory motor integration, and perception would ultimately lead to a better understanding of how the brain functions. Optical voltage-sensing probes are extremely useful in visualizing the complex interactions between neurons. Currently, many optical voltage-sensing probes are from the class of genetically encoded voltage indicators (GEVIs), where many are based on natural proteins. However, some of these probes are either too slow to measure the changes in membrane potential, too dim, or do not have sufficient changes in their relative fluorescence. We hypothesize that we will be able to design and construct GEVIs using maquettes, four-alpha-helical bundle synthetic proteins. When created, these maquettes will function as optical voltage-sensing probes that are bright, fast, and have large changes in their relative fluorescence. In our study, we will develop transmembrane maquette GEVIs based on two different mechanisms: the optical Stark effect and Förster resonance energy transfer (FRET). The first class of maquette GEVIs, based on the Stark effect, will comprise of a natural Stark effect cofactor coupled with an electron-transfer chain of hemes bound to the interior of the maquette. The natural Stark effect cofactor will experience a relative change in fluorescence as the membrane potential changes due to the electron transfer occurring within the maquette. The second class of maquette GEVIs will comprise of a fluorescent protein attached to a maquette with an electron-transfer chain of hemes. As the membrane potential changes, the fluorescence of the fluorescent protein will be quenched due to the change in redox states of the hemes. First, we will design sequences for each of the classes of maquette GEVIs. Using biophysical characterizations, such as testing the strength of cofactor binding sites and proper membrane insertion, we can determine the most successful candidates for a maquette GEVI. The electric field properties of the maquette GEVIs will be tested through different experiments. For the Stark effect maquettes, we will perform linear dichroism and Stark spectroscopy in order to obtain information on both the orientation of the Stark effect cofactors and the relative change in fluorescence. We will perform voltage-clamp fluorimetry across an artificial membrane on both the Stark effect maquettes and the maquette-fluorescent protein fusion constructs, so as to measure the field sensitivity of the maquette GEVIs as the membrane potential is changed.
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