Biophysical principles for the design of photoluminescent proteins

Abstract

Photoluminescence is the emission of light following optical excitation. As biological molecules typically have weak emission, photoluminescent compounds provide excellent analytical utility for interrogating biological systems. While synthetic dyes and labels are one way to provide the analytic signal for such interrogations, photoluminescent proteins offer the advantage of being categorically biocompatible. Photoluminescent proteins are also ideal targets for application-specific design, as their targeting to specific cellular loci, binding affinity for target analytes, or response to different environments may be modified while having minimal or predictable effect on the optical readout. This thesis analyses the biophysical underpinnings of how photoluminescent proteins may be designed to have the desired properties.

Following a general introduction in Chapter 1, Chapter 2 explores the design of phosphorescent proteins, pairing genetically encoded antennae and lanthanide binding sites for lanthanide (III) ions. This work presents a novel, stable, and high-yield lanthanide binding protein which exhibits nanomolar binding affinity. Chapter 3 characterises the tryptophan orientations within this designed protein with experimental electron-nuclear double resonance (ENDOR) spectroscopy. Analysis of ENDOR spectra reveals rotameric conformations that are absent from computationally predicted structures. Chapter 4 undergoes an in-depth analysis of the steric accommodation of 4-substituted tryptophans in maltose binding protein. This work shows the side-chain repacking that takes place to maintain the native global fold. Chapter 5 assesses the steric accommodation of benzadiazole-containing non-canonical amino acids, showing that the bulkier side chain is surprisingly isosteric for the canonical tryptophan. This work also presents experimental evidence for the expansion of the classical fluorescent protein catalysis to include a secondary amine between the aromatic side chain of the chromophore tripeptide and the backbone.

Finally, I conclude this thesis with summary of results, and areas of focus that would expand this current work.