Date of Award

Spring 5-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny Yang

Second Advisor

Osvaldo Delbono

Third Advisor

Giovanni Gadda

Fourth Advisor

Donald Hamelberg

Fifth Advisor

Mary Wagner

Abstract

Fluorescent protein based genetically encoded fluorescent reporters play an improtant role in understanding the cellular physiology by directly monitoring real-time cellular signaling pathways with fluorescent microscope.

Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a novel class of genetically encoded indicators by designing a Ca2+ binding site in the enhanced green fluorescent protein (EGFP). One of them, CatchER (Calcium sensor for detecting high concentration in the ER), exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s-1 and appropriate Ca2+ binding affinity, likely due to local, Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. CatchER reported considerable differences in ER Ca2+ dynamics and concentration among epithelial HeLa, kidney HEK 293, and muscle C2C12 cells, enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis (FDB) muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. Moreover, the structure of CatchER has been investigated by nuclear magnetic resonance spectroscope (NMR) and high-resolution X-ray crystal structures to understand the novel mechanism of Ca2+ induced fluorescent enhancement of GFP.

It is crucial to investigate the metal selectivity of Ca2+/Mg2+ of these metalloproteins to understand cellular physiology. The major Mg2+ binding sites of proteins have been reviewed and classified based on structural differences, and identified several key factors to determine Mg2+/Ca2+ selectivity with binding constants difference up to 104 in several types of metalloproteins.

Thrombin is involved in numerous cellular signaling pathways and plays a crucial role in blood coagulation. I designed a novel class of single EGFP-based thrombin sensors by inserting a thirty-amino acid short peptide with a thrombin cleavage site into the fluorescent sensitive location of EGFP. These designed protease sensors exhibited optimized kcat/Km up to 104 magnitudes higher than that of small peptide based absorption indicator EGR-pNA. The measured Km value is in below 10 mM, in the same magnitude as that of natural thrombin substrate Fibrinogen A.

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