Conformation dependence of pKa's of the chromophores from the purple asFP595 and yellow zFP538 fluorescent proteins (original) (raw)
Journal of The American Chemical Society, 2000
Green fluorescent proteins (GFPs) are being intensively investigated due to both their unusual optical spectroscopic characteristics and the extraordinary utility of GFPs as tools in biochemistry, cell biology, and molecular genetics. Recent studies have suggested that the spectrophotometric and fluorescence characteristics of GFPs are controlled through protonation states of the GFP chromophore (p-hydroxybenzylideneimidazolinone). However, of three protonation sites in the chromophore, only two have been studied. To understand the structural origin of the observed spectrophotometric and fluorescence characteristics of GFPs, employing ab initio methods, we have investigated all the possible protonation sites of the chromophore of denatured GFPs under different pH conditions. Our results suggest that the denatured GFP chromophore exists in not just two protonation states, as widely assumed in the literature, but in five different protonation states that depend on pH over the range -3.2 to 9.4 as assessed from the predicted pK a values and the self-consistent reaction field continuum calculations of solvation employing Schrödinger's Jaguar 3.5 program. The unexpected complexity of the protonation states of the denatured GFP chromophore postulated here may provide a useful starting point for a further investigation of the protonation states of the intact GFP chromophore responsible for the experimentally observed UV absorption and fluorescence emission properties of structurally intact GFPs.
Journal of the American Chemical Society, 2012
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.
Photoisomerization and proton transfer in photoactive yellow protein
Journal of the American Chemical Society, 2003
The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes. Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates. The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates. The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters. The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al. [Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature 1998, 392, 206-209] supports its assignment to the PYPB (I0) intermediate. Differences between the time-resolved room temperature (298 K) spectrum of the PYPB intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter. This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP L (I1 or pR) intermediate. The structure of the chromophore in the PYPL intermediate is determined computationally and shown to be deprotonated, in agreement with experiment. Calculations based on our PYP B and PYPL models lead to insights concerning the PYPBL intermediate, observed only at low temperature.
Photophysics of the blue fluorescent protein
Journal of Luminescence, 2007
The blue fluorescent protein (BFP) is a mutant of the green fluorescent protein, where the phenolic ring of the chromophore has been replaced by imidazole cycle of histidine residue. The usability of BFP as a fluorescent marker is hampered by its low fluorescence quantum yield at room temperature. The intensity of fluorescence increases by a factor of 4.5 when the temperature is decreased from 320 K down to 225 K. The fluorescence is also enhanced by hydrostatic pressure. Both effects have been explained by shift of the equilibrium between hydrogen nonbonded and hydrogen-bonded chromophores. Our semi-empirical quantum chemical calculations show that the fluorescence quantum yield of the BFP chromophore is low due to isomerization in the electronically excited state -twisting of the bridging bond by 901. At this twisted geometry the potential energy surfaces of ground and excited states are situated close to each other facilitating efficient nonradiative decay. r
Journal of Physical Chemistry B, 2008
We discuss the role of the protein in controlling the absorption spectra of photoactive yellow protein (PYP), the archetype xanthopsin photoreceptor, using quantum mechanics/molecular mechanics (QM/MM) methods based on ab initio multireference perturbation theory, combined with molecular dynamics (MD) simulations. It is shown that in order to get results in agreement with the experimental data, it is necessary to use a model that allows for a proper relaxation of the whole system and treats the states involved in the electronic spectrum in a balanced way, avoiding biased results due to the effect of nonrepresentative electrostatic interactions on the chromophore.
Chemical Physics Letters, 2004
The proton transfer from the green fluorescent protein chromophore to a nearby water molecule is studied by means of CASSCF, CASPT2 and TDDFT calculations. A 1 pr* electronic state is found to intersect with the photoactive 1 pp* electronic state along the proton transfer coordinate. This state crossing constitutes a possible non-radiative deactivation pathway of the photoexcited neutral form of the chromophore. A discussion on the performance of the different levels of theory employed is also given, focusing in the ability to correctly describe the 1 pr* electronic state.
Journal of …, 2003
Two ground-state protonation forms causing different absorption peaks of the green fluorescent protein chromophore were investigated by the quantum mechanical SAC/SAC-CI method with regard to the excitation energy, fluorescence energy, and ground-state stability. The environmental effect was taken into account by a continuum spherical cavity model. The first excited state, HOMO-LUMO excitation, has the largest transition moment and thus is thought to be the source of the absorption. The neutral and anionic forms were assigned to the protonation states for the experimental A-and B-forms, respectively. The present results support the previous experimental observations.
Photophysics and Spectroscopy of Fluorophores in the Green Fluorescent Protein Family
Abstract Proteins homologous to the green fluorescent protein (GFPs) form a large family of unconventional, genetically encoded fluorophores with widely diverse colors and applications, which have profoundly renewed the fields of biological imaging and drug screening. Their detailed spectroscopy stems from a complex interplay between the electronic properties of a relatively simple, yet flexible and multiprotonable chromophore formed after specific biosynthesis, and the spatial and dynamic organization of its protein carrier. Early experimental and theoretical studies of GFP from the Aequorea victoria jellyfish and of model synthetic compounds have revealed that chromophore twisting, cis-trans isomerization, proton transfer, and electron transfer are major excited state reactions that determine its photophysics and photochemistry. It has been found later that quite similar mechanisms are at work in several distant members of the GFP family, suggesting a unified picture that may guide the future development of new GFP-based biosensors. Graphical Abstract
Fluorescent proteins (FPs), featuring the same chromophore but different chromophoreprotein interactions, display remarkable spectral variations even when the same chromophore protonation state, i.e. the anionic state, is involved. We examine the mechanisms behind this tuning by means of structural analysis, molecular dynamics simulations, and vertical excitation energy calculations by QM/MM Time-Dependent Density Functional Theory (TD-DFT), CASPT2/CASSCF and SAC-CI. The proteins under investigation include the structurally similar, though spectrally distinct, Dronpa and mTFP0.7, with absorption peaks at 453 nm and 503 nm, respectively. We extend our analysis to two Green Fluorescent Protein variants, GFP-S65T (absorption peak at 484 nm), for comparison with previous computational studies, and GFP-S65G/V68L/S72A/T203Y, a Yellow Fluorescent Protein (514 nm), in order to include one of the most red shifted FPs containing a GFP-like chromophore. We compare different choices of the QM system, and we discuss how molecular dynamics simulations affect the calculation * To whom correspondence should be addressed of excitation energies, with respect to X-ray structures. We are able to partially reproduce the spectral tuning of the FPs and correlate it to the chromophore bond-length variations, as determined by specific interactions with the chromophore environment. ⊕ MD-snap(c1) (ave) © © © © ©