Rebekka M. Wachter

Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein.

BACKGROUND Because of its ability to spontaneously generate its own fluorophore, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria is used extensively as a fluorescent marker in molecular and cell biology. The yellow fluorescent proteins (YFPs) have the longest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-letter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T. RESULTS We have determined the crystal structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 A resolution, respectively. Both structures show clear electron density for nearly coplanar pi-pi stacking between Tyr203 and the chromophore. The chromophore has been displaced by nearly 1 A in comparison to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, intense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pKa values of 7.0 (YFP) and 8.0 (YFP H148G). CONCLUSIONS The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.

An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

Sensitivity of the yellow variant of green fluorescent protein to halides and nitrate

as those found in the hindbrain only occurred after 35 hpf (Figure 1e). In the hindbrain and the midbrain tegmentum, medio-laterally oriented spinning axes (that is, rotations in the observation plane) were predominant, whereas in the optic tectum, most nuclei did not spin around a medio-lateral axis — a difference that might be related to the different orientation of the neuroepithelium in that part of the brain. Because it is less accessible, the forebrain was not investigated. Interestingly, the neuroretina, although it originates from the brain neuroepithelium, did not have spinning nuclei at any stage of its development. A more extensive survey of the distribution and direction of nuclear rotations occurring in the observation plane was carried out in a well-defined compartment of the hindbrain, rhombomere 5, the limits of which can be fairly well discerned in the living embryo. Both clockwise and anticlockwise rotations were found throughout the rhombomere, with a somewhat reduced frequency anteriorly, both on the left and the right side of the rhombomere (Figure 1g). In the hindbrain or midbrain of the day-2 embryo, only a small minority of the cells of the neuroepithelium have already differentiated into neurons [1]. Although the larger, easily recognizable nuclei of these young neurons occasionally undergo partial or even complete rotations during a 1 hour recording session, this is by no means comparable to the high spin rates displayed by undifferentiated cells of the neuroepithelium. Only one report, by Pomerat in 1953 [2], concerning tissue cultures of adult human nasal mucosa, documented nuclei spinning at rates comparable to, although lower than, those reported here in the zebrafish embryo. Some later papers reported nuclear rotations in tissue cultures of neurons and in some cell lines, but at much lower rates (up to one rotation per hour, at most) [3–7]. The very high spinning rates found here in the zebrafish embryo occur only in the still undifferentiated brain neuroepithelium, and in a well defined developmental sequence — first in the hindbrain and subsequently in the midbrain. This observation raises at least three lines of questioning: how are the spinning nuclei anchored to the surrounding cell structures; what is the motive force that can make them spin so fast; and what is the possible functional significance of this phenomenon for the neuroepithelial cells involved? Supplementary material Supplementary material including time-lapse video recordings of the spinning nuclei shown in Figure 1 is available at …

Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants.

The maturation process of green fluorescent protein (GFP) entails a protein oxidation reaction triggered by spontaneous backbone condensation. The chromophore is generated by full conjugation of the Tyr66 phenolic group with the heterocycle, a process that requires C-H bond scission at the benzylic carbon. We have prepared isotope-enriched protein bearing tyrosine residues deuterated at the beta carbon, and have determined kinetic isotope effects (KIEs) on the GFP self-processing reaction. Progress curves for the production of H 2O 2 and the mature chromophore were analyzed by global curve fitting to a three-step mechanism describing preoxidation, oxidation and postoxidation events. Although a KIE for protein oxidation could not be discerned ( k H/ k D = 1.1 +/- 0.2), a full primary KIE of 5.9 (+/-2.8) was extracted for the postoxidation step. Therefore, the exocyclic carbon is not involved in the reduction of molecular oxygen. Rather, C-H bond cleavage proceeds from the oxidized cyclic imine form, and is the rate-limiting event of the final step. Substantial pH-dependence of maturation was observed upon substitution of the catalytic glutamate (E222Q), indicating an apparent p K a of 9.4 (+/-0.1) for the base catalyst. For this variant, a KIE of 5.8 (+/-0.4) was determined for the intrinsic time constant that is thought to describe the final step, as supported by ultra-high resolution mass spectrometric results. The data are consistent with general base catalysis of the postoxidation events yielding green color. Structural arguments suggest a mechanism in which the highly conserved Arg96 serves as catalytic base in proton abstraction from the Tyr66-derived beta carbon.

Mechanistic Diversity of Red Fluorescence Acquisition by GFP-like Proteins

This review aims to summarize our current state of knowledge of several post-translational modification mechanisms known to yield red fluorescence in the family of GFP-like (green fluorescent protein-like) proteins. We begin with a brief review of the maturation mechanism that leads to green fluorescence in GFPs. The main body of this article is focused on a series of main chain redox and beta-elimination reactions mediated by light and O(2), ultimately yielding a red-emitting chromophore. In all GFP-like proteins, a tyrosine-derived phenolic group constitutes an essential building block of the chromophores skeleton. Two major classes of red-emitting species have been identified in naturally occurring fluorescent proteins. In the DsRed type, an acylimine moiety is found to be conjugated to the GFP-like chromophore. Recent evidence has suggested that two mechanistic pathways, a green branch and a red branch, diverge from an early cyclic intermediate that bears a standard tyrosine side chain. Therefore, the long-standing notion that all FP colors originate from modifications of the GFP-like chromophore may need to be revised. In the Kaede-type green-to-red photoconvertible class of FPs, a light-mediated main chain elimination reaction partakes in the formation of a three-ring chromophore that involves the incorporation of a histidine residue into the conjugated system. A mechanistic role for photoexcitation of the GFP-like chromophore is undisputed; however, the nature of associated proton transfer steps and the charge state of the critical imidazole group remain controversial. In addition to the two major classes of red fluorescent proteins, we briefly describe yellow fluorescence arising from modifications of DsRed-type intermediates, and the less well understood photoactivated oxidative redding phenomenon.

The Family of GFP-Like Proteins: Structure, Function, Photophysics and Biosensor Applications. Introduction and Perspective

Abstract In this issue, we offer a symposium-in-print that is focused on several new advancements in fundamental research related to the family of GFP (green fluorescent protein)-like proteins. A few applied aspects are also included to illustrate the impact this amazing set of colored proteins has made on our understanding of cell biology at the molecular level. The six articles presented here cut across several disciplines ranging from biological function to protein structure to photophysical aspects. These highly original pieces of work include both experimental and computational approaches, and will provide the reader with significant insight into current, state-of-the-art research activities in this very dynamic and fast-paced field. In the first part of this perspective, I will give a brief overview of the history and salient features of GFPs, cite some examples that illustrate their impact on biotechnology, and provide a brief review of the structural and chemical features that lend these proteins their fascinating appearance. In the second part, I will introduce each of the peer-reviewed contributions of the participating authors.

Structural changes associated with the acute thermal instability of Rubisco activase

Inhibition of photosynthesis by heat has been linked to the instability of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) chaperone, Rubisco activase. Examination of the recombinant enzyme showed that ADP and ATP protected against inactivation, whereas Mg(2+) promoted inactivation. Heating caused aggregation of Rubisco activase characterized by disruption of secondary structure content and formation of insoluble protein. In contrast, incubation at room temperature without nucleotide caused the active approximately 660 kDa protein to form a soluble, but inactive aggregate of > 2 x 10(6) Da. Circular dichroism (CD) spectroscopy and fluorescence established that structural perturbations in the aggregate did not reduce alpha-helical content significantly. Differences in the thermal stability between wild type and mutant Rubisco activase were observed for the recombinant proteins and when the proteins were expressed in transgenic Arabidopsis. That the sensitivity of these plants to heat differs indicates that the thermal instability of Rubisco activase is a main determinant of the temperature-sensitivity of photosynthesis.

Atomic Resolution X-ray Structure of the Substrate Recognition Domain of Higher Plant Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

Background: Rubisco activase has been linked to the inhibition of net photosynthesis upon warming. Results: The structure of the C-terminal domain adopts an unusually elongated shape. Conclusions: Reactivation of Rubisco may involve movement of a paddle-like extension. Significance: This work will aid in gaining a better understanding of Rubisco regulation. The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO2 fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco–Rubisco activase interaction

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO2concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.

A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins.

In proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.