Synthetic Biology of Autofluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 96

Synthetic Biology of Autofluorescent Proteins
Michael Georg Hoesl, Lars Merkel, and Nediljko Budisa
Abstract Autofluorescent proteins (FPs), which to date are predominately used as
tools in cell biology and spectroscopy, have arrived in the focus of synthetic
biology. Thereby, the intention is to supplement classically used protein design
methods such as site-directed mutagenesis or guided evolution by expanding the
scope of protein synthesis. This is achieved by the co-translational introduction of
novel noncanonical amino acids (NCAAs) into proteins. In the following chapter,
we present current applications of an expanded amino acid repertoire for the design
of spectral and folding properties of FPs. We will show that NCAAs are not only
useful tools to study fundamental aspects of photophysics but also have great
potential to generate novel FP tools for cell biology applications. On the one
hand, aromatic amino acids other than the naturally occurring His, Tyr, Phe, and
Trp were used to create novel spectral classes of FPs by direct chromophore
modification. On the other hand, NCAAs were also applied for “FP protein matrix
engineering” to influence chromophore fluorescence and overall folding. We also
illustrate a practical application of these principles by presenting “golden annexin
A5” as a novel apoptosis detection tool designed by synthetic biology methods.
Finally, we describe a potential route to convert any protein of interest into a
chromo-protein by introduction of novel synthetic autofluorescent amino acids.
Keywords Chromophore variants, ECFP, EYFP, GFP, GFP structure, non-
canonical amino acid, synthetic biology
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
2 Chromophore Redesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
2.1 Chromophore with 4-Aminotryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
2.2 Spectroscopic Features of GdFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
2.3 Chromophore Halogenations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
M.G. Hoesl, L. Merkel, and N. Budisa ( * )
Department of Chemistry, Berlin Institute of Technology/TU Berlin, Biocatalysis Group,
Franklinstraße 29, 10587 Berlin, Germany
e-mail: [e-mail]