Crystallographic Characterization of Novel Glucose and Lactate Biosensors Pubblico

Abstract

Glucose metabolic pathways are potential therapeutic targets for epilepsy, Alzheimer’s disease, Huntington’s disease, and other neurological disorders. An intimate understanding of cerebral energy metabolism opens doors for the development of novel treatments, and obtaining this understanding requires advanced imaging techniques that intimately capture the activities of glucose and other energy metabolites in the mammalian brain. The Yellen Lab at Harvard Medical School designed two genetically encoded fluorescence lifetime-based biosensors, SweetieTS and LiLac, which allow for in vivo tracking of glucose and lactate, respectively. Upon ligand binding, each biosensor undergoes a conformation change that results in an observable change in fluorescence lifetime. Mutagenesis studies conducted in the Yellen lab offer insights into the mechanism of coupling and lifetime change in these biosensors; however, they also raise questions that require additional structural information to answer. Here, we used X-ray crystallography to solve the structures of SweetieTS and LiLac and corroborate our collaborators’ hypotheses. The two sub-2.5 Å crystal structures presented suggest that a linker between the two domains in each biosensor regulates solvent exchange near the chromophore. Ligand binding causes the linker to shift. This changes the amount of solvent quenching, which determines the lifetime of the biosensor. Though additional crystal structures of the biosensors in different states would be helpful in understanding these processes in more detail, this work continues an important conversation about the mechanism of lifetime change in these genetically encoded biosensors. Fluorescence lifetime imaging has been limited by the lack of efficient strategies to design lifetime biosensors: These insights will advance the development of new genetically encoded biosensors for fluorescence lifetime imaging.

Table of Contents

Introduction........................................................................................................................................ 1

Mapping Energy Metabolism in the Brain................................................................................................ 1

Genetically Encoded Fluorescent Indicators............................................................................................. 3

Practical Challenges & Solutions............................................................................................................. 6

Fluorescence-Lifetime Imaging: LiLac & SweetieTS.................................................................................. 9

Aim I.................................................................................................................................................... 11

Methods I............................................................................................................................................. 12

Protein Crystallization............................................................................................................................ 12

Protein X-ray Crystallography................................................................................................................. 15

Results I................................................................................................................................................ 16

Aim II.................................................................................................................................................... 23

Methods II............................................................................................................................................. 24

SweetieTS Mutants: Expression................................................................................................................ 24

SweetieTS Mutants: Lysis & Purification................................................................................................... 25

Protein X-ray Crystallography.................................................................................................................. 28

Results II............................................................................................................................................... 28

Analysis & Discussion........................................................................................................................... 29

Conclusion............................................................................................................................................ 38

References............................................................................................................................................. 40

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Parola chiave	FLIM imaging biosensors protein X-ray crystallography glucose metabolism
Committee Chair / Thesis Advisor	Davis, Katherine, Emory University
Committee Members	Dunham, Christine, Emory University Liu, Fang, Emory University

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