Fitness landscapes1,2, depictions of how genotypes manifest at the phenotypic level, form the basis for our understanding of many areas of biology2–7 yet their properties remain elusive. Studies addressing this issue often consider specific genes and their function as proxy for fitness2,4, experimentally assessing the impact on function of single mutations and their combinations in a specific sequence2,5,8–15 or in different sequences2,3,5,16–18. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here, we chart an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function, fluorescence, of tens of thousands of derivative genotypes of avGFP. We find that its fitness landscape is narrow, with half of genotypes with two mutations showing reduced fluorescence and half of genotypes with five mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations arising mostly through the cumulative impact of slightly deleterious mutations causing a threshold-like decrease of protein stability and concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for a number of fields including molecular evolution, population genetics and protein design.

Members of the green fluorescent protein (GFP) family form chromophores by modifications of three internal amino acid residues. Previously, many key characteristics of chromophores were studied using model compounds. However, no studies of intermolecular excited-state proton transfer (ESPT) with GFP-like synthetic chromophores have been performed because they either are nonfluorescent or lack an ionizable OH group. In this paper we report the synthesis and photochemical study of two highly fluorescent GFP chromophore analogues: p-HOBDI-BF2 and p-HOPyDI:Zn. Among known fluorescent compounds, p-HOBDI-BF(2) is the closest analogue of the native GFP chromophore. These irrreversibly (p-HOBDI-BF(2)) and reversibly (p-HOPyDI:Zn) locked compounds are the first examples of fully planar GFP chromophores, in which photoisomerization-induced deactivation is suppressed and protolytic photodissociation is observed. The photophysical behavior of p-HOBDI-BF2 and p-HOPyDI:Zn (excited state pK(a)'s, solvatochromism, kinetics, and thermodynamics of proton transfer) reveals their high photoacidity, which makes them good models of intermolecular ESPT in fluorescent proteins. Moreover, p-HOPyDI:Zn is a first example of "super" photoacidity in metal-organic complexes.

Superphotoacidity involves ultrafast proton motions implicated in numerous chemical and biological processes. We used conformational locking and strategic addition of electron-withdrawing substituents to synthesize a new GFP chromophore analogue: p-HO-3,5-diF-BDI:BF (diF). It is highly fluorescent and exhibits excited-state proton transfer (ESPT) in various solvents, placing it among the strongest photoacids. Tunable femtosecond stimulated Raman spectroscopy with unique resonance conditions and transient absorption are complementarily employed to elucidate the structural basis for superphotoacidity. We reveal a multistep ESPT reaction from diF to methanol with an initial proton dissociation on the ∼600 fs time scale that forms a charge-separated state, stabilized by solvation, and followed by a diffusion-controlled proton transfer on the ∼350 ps time scale. A ∼1580 cm phenolic ring motion is uncovered to accompany ESPT before 1 ps. This study provides a vivid movie of the photoinduced proton dissociation of a superphotoacid with bright fluorescence, effectively bridging fundamental mechanistic insights to precise control of macroscopic functions.

Study of fungal bioluminescence mechanisms generates development of a multicolor enzymatic chemiluminescence system.

A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red-shifted spectra, high fluorescence quantum yields and pH-independence in physiological pH range. The intra- and intermolecular mechanisms of radiationless deactivation of ABDI-BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with "infinite" photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

A novel method of protein labeling uses the highly dynamic reversible association of a cell-permeable fluorogenic dye and lipocalin Blc mutants.

We devise a unique “double-donor–one-acceptor” strategy to effectively tune the “dark” GFP-core outside protein matrix into redder and brighter fluorophores.