Fluorescent Proteins I

Fluorescence is a photophysical phenomenon, which obeys basic physical laws. The fluorescence of the autofluorescent proteins arises on the molecular level from chromophores, which are buried in the protein matrix. The three-dimensional, well-defined architecture of the surrounding is a prerequisite for their function. Excitation of the isolated chromophores leads only to a negligible light emission at room temperature. Several processes competing with the radiative decay are responsible for the quenching. To understand how nature has learned to suppress these alternative pathways from the excited state in autofluorescent proteins, the molecular dynamics as well as the influence of several amino acids in the interior of the protein has to be analysed. We review the current status of the understanding of the non-radiative decay mechanisms for the different fluorescent protein classes, i.e., colours. Furthermore, we address what can be learned from fluorescence lifetime measurements and how they can be exploited for analytical purposes such as fluorescence lifetime imaging microscopy. Finally, we sketch the needs of increased fluorescence quantum yields and present strategies to prolong the fluorescence lifetimes.

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.

Green fluorescence protein (GFP) wild type and some of its mutants undergo excited state proton transfer between the chromophore and the nearby Glu222 residue. This process has been covered in detail in the chapter written by Stephen Meech. Apart from this ultrafast photochemical reaction, multiple other proton-transfer processes take place in the GFP protein matrix, and these will be covered in this chapter. For example, proton exchange between the chromophore and the nearby bulk solvent may occur via His148 that is located in hydrogen-bonding distance from the chromophore and provides direct access to the bulk solvent. Moreover, two extended proton-transfer wires including titratable residues as well as a number of buried water molecules connect the chromophore to the protein surface. Based on a recent high-resolution X-ray structure of GFP, all titratable groups of the protein could be placed in one of these two large hydrogen-bonding clusters, suggesting that a multitude of proton-transfer processes can occur in the GFP matrix at any moment in time. While it is quite likely that similar proton pathways also exist in other soluble and membrane proteins, they are much harder to study. GFP is an exciting model system for monitoring those processes as they often directly affect the chromophore photophysics. The dynamics of proton exchange inside the GFP barrel and with bulk solvent has thus been characterized by fluorescence correlation spectroscopy (FCS) of the chromophore fluorescence and by pH-jump experiments. These studies showed that the autocorrelation of the chromophore fluorescence is affected either by pH-independent processes on microsecond to millisecond time scales or by pH-dependent processes on similar time scales. The former ones are likely proton equilibria occurring within the GFP barrel, and the latter ones are likely exchange processes with the solvent. Biomolecular simulation methods are now being developed, which will soon allow accessing such time scales by computational means. Then, we will hopefully be able to connect the spectroscopic findings with dynamic atomistic simulations of proton-transfer dynamics.

GFP-like proteins, originally cloned from marine animals, are genetically encoded fluorescence markers that have become indispensable tools for the life sciences. The search for GFP-like proteins with novel and improved properties is ongoing, driven by the persistent need for advanced and specialized fluorescence labels for cellular imaging. The 3D structures of these proteins are overall similar. However, considerable variations have been found in the covalent structures and the stereochemistry of the chromophore, which govern essential optical properties such as the absorption/emission wavelengths. A detailed understanding of the structure and dynamics of GFP-like proteins greatly aids in the rational engineering of advanced fluorescence marker proteins. In this chapter, we summarize the present knowledge of the structural diversity of GFP-like proteins and discuss how structure and dynamics govern their optical properties.