Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry.

Red fluorescent indicators for calcium ion (Ca(2+)) are preferable, relative to blue-shifted alternatives, for biological imaging applications due to the lower phototoxicity, lower autofluorescent background and deeper tissue penetration associated with longer wavelength light. Accordingly, we undertook the development of a genetically encoded Ca(2+) indicator based on the popular and widely utilized Discosoma-derived red fluorescent protein, mCherry. Starting from a promising but dimly fluorescent circular permutated variant of mCherry, we first engineered a 13-fold brighter variant (cp196V1.2) through directed evolution. This bright cp196V1.2 was then used as the scaffold for creation of eight distinct libraries of potential Ca(2+) indicators via permutation at different sites within the 7th and 10th ß-strands, and fusion of calmodulin and M13 to the new termini. Screening of these libraries led to the conclusion that, consistent with previous investigations of homologous fluorescent proteins, the 146-145 site in ß-strand 7 is the most promising permutation site for construction of useful Ca(2+) indicators. Further rounds of directed evolution ultimately led to an indicator that exhibits a 250% change in intrinsic brightness in response to Ca(2+) and an exceptionally high affinity (Kd = 6 nM) for Ca(2+).

Mutational analysis of a red fluorescent protein-based calcium ion indicator.

As part of an ongoing effort to develop genetically encoded calcium ion (Ca2+) indicators we recently described a new variant, designated CH-GECO2.1, that is a genetic chimera of the red fluorescent protein (FP) mCherry, calmodulin (CaM), and a peptide that binds to Ca2+-bound CaM. In contrast to the closely related Ca2+ indicator R-GECO1, CH-GECO2.1 is characterized by a much higher affinity for Ca2+ and a sensing mechanism that does not involve direct modulation of the chromophore pKa. To probe the structural basis underlying the differences between CH-GECO2.1 and R-GECO1, and to gain a better understanding of the mechanism of CH-GECO2.1, we have constructed, purified, and characterized a large number of variants with strategic amino acid substitutions. This effort led us to identify Gln163 as the key residue involved in the conformational change that transduces the Ca2+ binding event into a change in the chromophore environment. In addition, we demonstrate that many of the substitutions that differentiate CH-GECO2.1 and R-GECO1 have little influence on both the Kd for Ca2+ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.

Circularly permuted monomeric red fluorescent proteins with new termini in the beta-sheet.

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca(2+) and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole-dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th beta-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca(2+) biosensors.

Genetically encoded FRET-based biosensors for multiparameter fluorescence imaging.

The phenomenon of Förster (or fluorescence) resonance energy transfer (FRET) between two fluorescent proteins of different hues provides a robust foundation for the design and construction of biosensors for the detection of intracellular events. Accordingly, FRET-based biosensors for a variety of biologically relevant ions, molecules, and specific enzymatic activities, have now been developed and used to investigate numerous questions in cell biology. An emerging trend in the use of FRET-based biosensors is to apply them in combination with a second biosensor in order to achieve simultaneous imaging of multiple biochemical parameters in a single living cell. Here we discuss the particular technological challenges facing the use of FRET-based biosensors in multiparameter live cell fluorescence imaging and highlight recent efforts to overcome these challenges. In addition, we survey recent applications and provide an outlook on the future opportunities in this area.

Computational prediction of absorbance maxima for a structurally diverse series of engineered green fluorescent protein chromophores.

By virtue of its self-sufficiency to form a visible wavelength chromophore within the confines of its tertiary structure, the Aequorea victoria green fluorescent protein (GFP) is single-handedly responsible for the ever-growing popularity of fluorescence imaging of recombinant fusion proteins in biological research. Engineered variants of GFP with altered excitation or emission wavelength maxima have helped to expand the range of applications of GFP. The engineering of the GFP variants is usually done empirically by genetic modifications of the chromophore structure and/or its environment in order to find variants with new photophysical properties. The process of identifying improved variants could be greatly facilitated if augmented or guided by computational studies of the chromophore ground and excited-state properties and dynamics. In pursuit of this goal, we now report a thorough investigation of computational methods for prediction of the absorbance maxima for an experimentally validated series of engineered GFP chromophore analogues. The experimental dataset is composed of absorption maxima for 10 chemically distinct GFP chromophore analogues, including a previously unreported Y66D variant, measured under identical denaturing conditions. For each chromophore analogue, excitation energies and oscillator strengths were calculated using configuration interaction with single excitations (CIS), CIS with perturbative correction for double substitutions [CIS(D)], and time-dependent density functional theory (TD DFT) using several density functionals with solvent effects included using a polarizable continuum model. Comparison of the experimental and computational results show generally poor quantitative agreement with all methods attempted. However, good linear correlations between the calculated and experimental excitation energies (R2>0.9) could be obtained. Oscillator strengths obtained with TD DFT using pure density functionals also correlate well with the experimental values. Interestingly, most of the computational methods used in this work fail in the case of nonaromatic Y66S and Y66L protein chromophores, which may be related to a significant contribution of double excitations to their excited-state wavefunctions. These results provide an important benchmark of the reliability of the computational methods as applied to GFP chromophore analogues and lays a foundation for the computational design of GFP variants with improved properties for use in biological imaging.