New directions in biological imaging: Engineering, characterization, and discovery of LOV-based fluorescent proteins

flavin-based fluorescent proteins;Light-oxygen-voltage sensing (LOV) domains;directed evolution;genome mining

Mukherjee, Arnab

关键词: flavin-based fluorescent proteins;    Light-oxygen-voltage sensing (LOV) domains;    directed evolution;    genome mining;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/49539/Arnab_Mukherjee.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In this work, I describe the characterization, engineering, and discovery of a new class of fluorescent reporters based on flavin-binding domains of light, oxygen, and voltage (LOV) sensing photoreceptor proteins. Flavin-based fluorescent proteins (FbFPs) are characterized by oxygen-independent maturation of fluorescence, which is a significant advantage compared to widely used fluorescent probes based on the green fluorescent protein (GFP) that are strictly dependent on oxygen for fluorescence. Broad application of FbFPs, has however, been hindered by an incomplete understanding of their performance and properties as viable fluorescent tags and by low levels of brightness of the existing set of FbFPs. In this work, I systematically addressed these issues with a view towards enabling pervasive application of FbFPs as a new set of fluorescent tags. First, I extensively characterized key biochemical and biophysical properties of existing FbFPs and demonstrated that aside from oxygen-independent fluorescence, FbFPs also exhibit rapid maturation of fluorescence (T1/2 < 2 min.), thermal stability (up to 60 °C), and a broad operational pH range (pH 4-11). Next, based on an improved understanding of FbFPs, I used directed evolution via site saturation mutagenesis to engineer 2-fold brighter mutants of an FbFP — F37S and F37T PpFbFP. Finally, I developed and applied a powerful approach based on genome mining to discover two new FbFPs from the fresh-water algae — Chlamydomonas reinhardtii and Vaucheria frigida (CreiLOV and VafLOV). Strikingly, CreiLOV emerged as the brightest known member of the FbFP library in addition to embodying several advantages in a single FbFP variant including a small size, monomeric form, robust photostability, broad operational pH and temperature range. Furthermore, I validated the application of FbFPs as transcriptional reporters for monitoring dynamic gene expression in Escherichia coli. FbFPs are at an early stage of development and their application as fluorescent tags for biological studies has only recently been pursued. From this perspective, my work presents a valuable framework to develop the emerging family of FbFPs, thereby potentially extending fluorescence imaging to an exciting class of biological systems that is intractable to GFP-based imaging (e.g., gene regulation in extremophiles, anaerobic pathogenesis, high density fermentations, hypoxic solid tumors, and human gastrointestinal microbiota).