Fluorescence and photoisomerization in photoactive proteins
Abstract/Contents
- Abstract
- Energetics features govern all physical processes. This dissertation examines how energetics features, namely electronic excited-state energy barriers and conical intersection branching ratios, affect fluorescence and photoisomerization in two model systems: split green fluorescent proteins (GFPs) and photoactive yellow protein (PYP). Chapter One frames the concept of energetics features in the broader context of rational protein design. Rational protein design, though yet in its nascent stages, is a field with great opportunity to shape the world in which we live by allowing for creation of purposefully designed enzymes that catalyze reactions of interest at high rates and efficiencies. A fundamental requirement for rational design is understanding how protein environments shape energetics features along reaction coordinates for processes they catalyze, photophysical and otherwise. Chapter Two examines the mechanism and bottlenecks of strand photodissociation in split GFPs. Split GFPs contain two or more constituent parts that only fluoresce on complementing with one another. Although this complementation is typically irreversible, light accelerates dissociation of a noncovalently attached b-strand from a circularly permuted split GFP, allowing the interaction to be reversible. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics--function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Because no previous projects in the Boxer lab have investigated PYP, Chapter Three provides a brief overview of PYP's history and basic photophysical properties. Chapter Four examines photoisomerization in PYP as part of an ongoing collaboration to test de novo protein design in a model system. In de novo protein design, computers use physical principles and empirical observations to produce proteins with desired functions that have sequences unrelated to those found in nature. In order to evaluate the prospect of predicting relative photophysical function in proteins of known structure and function, we proposed predicting the relative photoisomerization quantum yields of a range of point mutants in the PYP model system and then comparing those predictions to experimentally determined yields. Experimental results reveal that photophysical properties in PYP are weighted averages of quantum yields for conformers from heterogeneous PYP ground states and are highly sensitive to the relative populations of the individual conformers. Our observations indicate that accurately predicting a given mutant's relative photoisomerization quantum yield requires understanding that mutant's conformational heterogeneity and the photophysical properties of each of its conformers.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Both, Johan Hendrik Christiaan |
|---|---|
| Degree supervisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Du Bois, Justin |
| Thesis advisor | Martinez, Todd J. (Todd Joseph), 1968- |
| Degree committee member | Du Bois, Justin |
| Degree committee member | Martinez, Todd J. (Todd Joseph), 1968- |
| Associated with | Stanford University, Department of Chemistry. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Johan Hendrik Christiaan Both. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by Johan Hendrik Both
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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