Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems (227 references). “If wood is rubbed with the Pulmo marinus, it will have all the appearance of being on fire; so much so, indeed, that a walking-stick, thus treated, will light the way like a torch” (translation of Pliny the Elder from John Bostock, 1855).

The fluorescent protein palette: tools for cellular imaging

Cyan variants of green fluorescent protein (CFPs) are widely used as donors in FRET experiments. Here, a new CFP, mTurquoise2, is developed, which displays a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime.

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Structure-Guided Evolution of Cyan Fluorescent Proteins Towards a Quantum Yield of 93%

Rational protein engineering requires a holistic understanding of protein function. Here, we apply deep learning to unlabeled amino-acid sequences to distill the fundamental features of a protein into a statistical representation that is semantically rich and structurally, evolutionarily and biophysically grounded. We show that the simplest models built on top of this unified representation (UniRep) are broadly applicable and generalize to unseen regions of sequence space. Our data-driven approach predicts the stability of natural and de novo designed proteins, and the quantitative function of molecularly diverse mutants, competitively with the state-of-the-art methods. UniRep further enables two orders of magnitude efficiency improvement in a protein engineering task. UniRep is a versatile summary of fundamental protein features that can be applied across protein engineering informatics.

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Unified rational protein engineering with sequence-based deep representation learning

Phylogenetic inference from sequences can be misled by both sampling (stochastic) error and systematic error (nonhistorical signals where reality differs from our simplified models). A recent study of eight yeast species using 106 concatenated genes from complete genomes showed that even small internal edges of a tree received 100% bootstrap support. This effective negation of stochastic error from large data sets is important, but longer sequences exacerbate the potential for biases (systematic error) to be positively misleading. Indeed, when we analyzed the same data set using minimum evolution optimality criteria, an alternative tree received 100% bootstrap support. We identified a compositional bias as responsible for this inconsistency and showed that it is reduced effectively by coding the nucleotides as purines and pyrimidines (RY-coding), reinforcing the original tree. Thus, a comprehensive exploration of potential systematic biases is still required, even though genome-scale data sets greatly reduce sampling error.

Genome-scale phylogeny and the detection of systematic biases

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.

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Diversity and evolution of coral fluorescent proteins

Here we investigate the evolutionary scenarios that led to the appearance of fluorescent color diversity in reef-building corals. We show that the mutations that have been responsible for the generation of new cyan and red phenotypes from the ancestral green were fixed with the help of positive natural selection. This fact strongly suggests that the color diversity is a product of adaptive evolution. An unexpected finding was a set of residues arranged as an intermolecular binding interface, which was also identified as a target of positive selection but is nevertheless not related to color diversification. We hypothesize that multicolored fluorescent proteins evolved as part of a mechanism regulating the relationships between the coral and its algal endosymbionts (zooxanthellae). We envision that the effect of the proteins' fluorescence on algal physiology may be achieved not only through photosynthesis modulation, but also through regulatory photosensors analogous to phytochromes and cryptochromes of higher plants. Such a regulation would require relatively subtle, but spectrally precise, modifications of the light field. Evolution of such a mechanism would explain both the adaptive diversification of colors and the coevolutionary chase at the putative algae-protein binding interface in coral fluorescent proteins.

Adaptive Evolution of Multicolored Fluorescent Proteins in Reef-Building Corals

Proteins of the green fluorescent protein family represent a convenient experimental model to study evolution of novelty at the molecular level. Here, we focus on the origin of Kaede-like red fluorescent proteins characteristic of the corals of the Faviina suborder. We demonstrate, using an original approach involving resurrection and analysis of the library of possible evolutionary intermediates, that it takes on the order of 12 mutations, some of which strongly interact epistatically, to fully recapitulate the evolution of a red fluorescent phenotype from the ancestral green. Five of the identified mutations would not have been found without the help of ancestral reconstruction, because the corresponding site states are shared between extant red and green proteins due to their recent descent from a dual-function common ancestor. Seven of the 12 mutations affect residues that are not in close contact with the chromophore and thus must exert their effect indirectly through adjustments of the overall protein fold; the relevance of these mutations could not have been anticipated from the purely theoretical analysis of the protein's structure. Our results introduce a powerful experimental approach for comparative analysis of functional specificity in protein families even in the cases of pronounced epistasis, provide foundation for the detailed studies of evolutionary trajectories leading to novelty and complexity, and will help rational modification of existing fluorescent labels.

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Retracing Evolution of Red Fluorescence in GFP-Like Proteins from Faviina Corals

Resurrecting ancestral proteins in the laboratory can be a powerful tool in studies of protein structure and function as they can offer a rare glimpse into the evolutionary history of molecular function (Malcolm et al., 1990; Adey et al., 1994; Chandrasekharan et al., 1996; Dean and Golding, 1997; Bishop et al., 2000; Chang and Donoghue, 2000; Sun et al., 2002; Zhang and Rosenberg, 2002; Thornton, 2004). Another, perhaps even more intriguing reason for reconstructing ancestral proteins lies in the hope of achieving a better understanding of the biology of ancient animals that may have possessed these proteins (Jermann et al., 1995; Messier and Stewart, 1997; Galtier et al., 1999; Benner, 2002; Gaucher et al., 2003). Proteins that are involved in sensory systems might be particularly revealing with respect to the physiology and behavior of ancient animals that can no longer be studied directly in the laboratory (Nei et al., 1997; Boissinot et al., 1998; Chang et al., 2002). Moreover, experimental tests of laboratory-recreated ancestral proteins would provide information different from that obtained through studies of fossils. Although interpretations based on recreations of single molecules are of course limited, under the best of circumstances one may hope to test some of the theories of ancient animal biology derived from other methods such as paleontological studies (Chang et al., 2002). Reconstructions of the past depend entirely on the accuracies and limitations of the statistical methods and models employed. However, even in cases of deep divergences, when the accuracy of the reconstruction may be low, the experimental outcome remains valuable, as the effects of altering specific amino acids on a protein’s structure and function can be interesting, independently of whether or not the sequence represents the true ancestor. The general approach of using site-directed mutagenesis methods to alter amino acids in order to assess shifts in function is one of the most widely employed in studying protein function. Advances in phylogenetic methods of ancestral reconstruction, particularly the development of likelihood/Bayesian models that incorporate many different aspects of sequence evolution, have led to a plethora of models and methods available for use in phylogenetic reconstruction in recent years (Thorne, 2000; Huelsenbeck and Bollback, 2001; Whelan et al., 2001; Nielsen, 2005). This chapter briefly discusses some of the models available for use in ancestral reconstruction, then describes ways to address variation in reconstructed ancestral sequences when the intent is to recreate proteins

Dealing with model uncertainty in reconstructing ancestral proteins in the laboratory: examples from archosaur visual pigments and coral fluorescent proteins

Steven F. Field

Papers

Diversity and evolution of coral fluorescent proteins

Adaptive Evolution of Multicolored Fluorescent Proteins in Reef-Building Corals

Retracing Evolution of Red Fluorescence in GFP-Like Proteins from Faviina Corals

Dealing with model uncertainty in reconstructing ancestral proteins in the laboratory: examples from archosaur visual pigments and coral fluorescent proteins