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The development of novel genetically encoded voltage indicators using de novo designed proteins / Martin Joshua Iwanicki.
- Format:
- Book
- Thesis/Dissertation
- Author/Creator:
- Iwanicki, Martin Joshua, author.
- Language:
- English
- Subjects (All):
- Biophysics.
- Biochemistry.
- Plasma.
- Mutation.
- Potassium.
- Mutagenesis.
- Proteins.
- Membranes.
- Neurons.
- Oxidation.
- Phosphatase.
- Permeability.
- Nervous system.
- Biochemistry and Molecular Biophysics--Penn dissertations.
- Penn dissertations--Biochemistry and Molecular Biophysics.
- Local Subjects:
- Biophysics.
- Biochemistry.
- Plasma.
- Mutation.
- Potassium.
- Mutagenesis.
- Proteins.
- Membranes.
- Neurons.
- Oxidation.
- Phosphatase.
- Permeability.
- Nervous system.
- Biochemistry and Molecular Biophysics--Penn dissertations.
- Penn dissertations--Biochemistry and Molecular Biophysics.
- Genre:
- Academic theses.
- Physical Description:
- 1 online resource (174 pages)
- Contained In:
- Dissertations Abstracts International 83-03B.
- Place of Publication:
- [Philadelphia, Pennsylvania] : University of Pennsylvania ; Ann Arbor : ProQuest Dissertations & Theses, 2021.
- Language Note:
- English
- System Details:
- Mode of access: World Wide Web.
- text file
- Summary:
- Developing optical probes that can monitor voltage signaling events on the sub-microsecond timescale and can be targeted to specific cells and membranes is crucial to advance our understanding of the communication between neurons. Organic voltage sensitive dyes can report changes in membrane potential on appropriate timescales with high sensitivity; however, they cannot be genetically targeted to specific cell types. This lack of targetability is solved by using genetically encoded voltage indicators (GEVIs). GEVIs are typically constructed from the voltage-sensing domain of natural proteins, but they tend to be dimmer and slower than organic voltage sensitive dyes. Here, we present our progress on the development and characterization of a new family of genetically encoded voltage indicators called THOR (Transmembrane Hemoprotein Optical Reporter), a fusion protein of a de novo designed transmembrane 4-α-helical protein and a fluorescent optical reporter. THORs bind heme in the core of the 4-α-helical bundle, and the heme oxidation states change as a function of the transmembrane electric field. The oxidation state of one of the heme cofactors is then reported via FRET by an attached fluorescent protein. The speed of THORs is dependent on electron transfer between the cofactors, and therefore, tunable by their distance. Based on the Moser-Dutton Ruler of electron transfer, we can achieve electron transfer rates on the µs timescale between heme cofactors. Water-soluble prototypes of THOR expressed in E. coli revealed an 18% fluorescent quenching of mOrange2 and a 57% fluorescent quenching of Sirius upon heme reduction. Several different variants of THOR were designed with two different lengths: one that closely matches the thickness of the membrane and one that extends into the cytoplasm. THORs have been expressed in HEK293t cells, rat hippocampal neurons, and mouse primary neurons. The trafficking of THORs into the plasma membrane was improved with the sequential addition of export tag sequences from the potassium ion channel, Kir2.1. THORs expressed in neurons exhibit a fluorescence decrease in response to a potassium ion-induced depolarization. This work presents the development of the first de novo designed GEVI for improved faster sensing of neuronal electrical events.
- Notes:
- Source: Dissertations Abstracts International, Volume: 83-03, Section: B.
- Advisors: Discher, Bohdana M.; Committee members: Petersson, E. James; Anna, Jessica M.; Murakami, Kenji; Solomon, Lee A.
- Department: Biochemistry and Molecular Biophysics.
- Ph.D. University of Pennsylvania 2021.
- Local Notes:
- School code: 0175
- ISBN:
- 9798535571232
- Access Restriction:
- Restricted for use by site license.
- This item is not available from ProQuest Dissertations & Theses.
- This item must not be sold to any third party vendors.
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