Properties Of Green Fluorescent Protein Research Articles

Modulating the pH dependent photophysical properties of green fluorescent protein.

The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-l-tyrosine (mNO2Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the pK a of the 3-nitrophenol side chain of mNO2Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the pK a of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.

Ultrafast excited state relaxation of a model green fluorescent protein chromophore: Femtosecond fluorescence and transient absorption study

The intrinsic excited state properties of green fluorescent protein (GFP) chromophore have inspired syntheses of its structural analogues with a motivation to replicate or improve its excited state properties for biological imaging. In this work, we report torsional-motion mediated multi-coordinate relaxation pathway to explain the excited state dynamics of a GFP chromophore analogue, (4Z)-4-(4-N,N-Dimethylaminobenzylidene)-1,2-diphenyl-1,4-dihydro-5H-imidazolin-5-one (DPI), using femtosecond fluorescence up-conversion, femtosecond transient absorption and quantum chemical calculations. The excited state dynamics of this chromophore involves the formation of an N,N-disubstituted amine twisting mediated intramolecular charge transfer state on a sub-picosecond timescale. This intramolecular charge transfer event prepares an intermediate state that undergoes a “non-volume conserving” twist motion along an exocyclic double bond. The quantum chemical computations further predict that the twist motion along the exocyclic double bond is activated and leads to the formation of a conical intersection between S1 and S0 states of DPI, which is the main nonradiative channel of deactivation. This study confirms that for a structural analogue of GFP chromophore to exhibit excited state properties like the GFP, would require preventing such nonradiative pathways, which can be achieved by restricting the twisting degree of freedom about the exocyclic double bond.

The dynamic surface properties of green fluorescent protein and its mixtures with poly(N,N-diallyl-N-hexyl-N-methylammonium chloride)

Measurements of the dynamic surface elasticity and surface pressure of solutions of green fluorescent protein (GFP) indicate the formation of a dense protein monolayer with a relatively high surface elasticity of approximately 65 mN/m, and are typical for solutions of globular proteins. The mixed solutions of GFP and poly(N,N-diallyl-N-hexyl-N-methylammonium chloride) (PDAHMAC) demonstrate more complex behavior. During the first adsorption step the surface properties are determined by an unbound component, while the next step is mainly the adsorption of the complexes. The dynamic surface elasticity at relatively low polyelectrolyte concentrations (< 2*10−4 g/l) is close to the values for pure protein solutions when the surface pressure is less than 15 mN/m. At higher surface pressures the adsorption of complexes results in an increase of the surface elasticity up to 80 mN/m. If PDAHMAC concentrations are higher than 2*10−4 g/l, the kinetic dependences of the dynamic surface elasticity become non-monotonic and this quantity does not exceed 45 mN/m. The local maximum of the surface elasticity is a result of the formation of the distal region of the surface layer by the polyelectrolyte. Beyond the subsequent local minimum the surface elasticity starts to increase again due to the adsorption of GFP/ PDAHMAC complexes.

Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

Green fluorescent proteins (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting that all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all of the observed strong correlations among photophysical properties; related subtopics are extensively discussed in the Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating the photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue that this model should also be generally applicable to both biological and nonbiological polymethine dyes.

Utilizing Tyrosine Analogs to Alter Photophysical Properties of Green Fluorescent Protein

Utilizing Tyrosine Analogs to Alter Photophysical Properties of Green Fluorescent Protein

Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins.

Green fluorescent proteins (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting that all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all of the observed strong correlations among photophysical properties; related subtopics are extensively discussed in the Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating the photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue that this model should also be generally applicable to both biological and nonbiological polymethine dyes.

Endo- and Exo-Functionalized Tetraphenylethylene M12L24 Nanospheres: Fluorescence Emission inside a Confined Space.

The intrinsic relationship between the properties of green fluorescent protein (GFP) and its encapsulated small molecular light machine has spurred many biomimicking studies, aiming at revealing the detailed mechanism and further promoting its wide applications in different disciplines. However, how to build a similar confined microenvironment to mimic the cavity of a β-barrel and the fluorescence turn-on process is a fundamental challenge for both chemists and biologists. Herein, two distinct exo- and endo-functionalized tetraphenylethylene (TPE)-based M12L24 nanospheres with precise distribution of anchored TPE moieties and unique photophysical properties were constructed by means of a coordination-driven self-assembly strategy. Under dilute conditions, the nanospheres fluoresce more strongly than the corresponding TPE subcomponents. Meanwhile, the endo-functionalized sphere is able to induce a higher local concentration and more restrained motion of the enclosed 24 TPE units compared with exo-functionalized counterpart and thus induces much stronger emission due to the restriction of the rotation of the pendant TPE units. The biomimetic methodology developed here represents a promising way to understand and construct artificial GFP materials on the platforms of supramolecular coordination complexes.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Using Green Fluorescent Protein to Correlate Temperature and Fluorescence Intensity into Bacterial Systems

The unique and stunning spectroscopic properties of Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria, not to mention of its remarkable structural stability, have made it one of the most widely studied and used molecular tool in medicine, biochemistry, and cell biology. Its high fluorescent quantum yield is due to its chromophore, structure responsible of emitting green visible light when excited at 395 nm. Although it is noteworthy that there is enormous available information of the wonderful luminescent properties of GFP, the fact is that there are features and properties unexplored yet, particulary about its capabilities as molecular reporter in several biological processes. In this work, we used recombinant DNA technology to express the protein in bacteria; prepared the bacterial system both in liquid and solid media, and assembled an experimental set to expose those media to a laser beam; thereby we excited the protein chromophore and used emission spectroscopy in order to observe variations in fluorescence when the bacterial system is exposed to different temperatures.

Graphene Oxide‐mediated Fluorescence Quenching of Green Fluorescent Protein for Biomedical Applications

We investigated the fluorescence property of green fluorescent protein (GFP) with graphene oxide (GO) for both non‐covalent and covalent interactions. The mechanism of GO‐mediated quenching of GFP fluorescence is the combination of both static and dynamic quenching. Although GFP exhibits two absorption bands with similar fluorescence emission spectra, the quenching efficiency of the two absorption bands from non‐covalent immobilization were different—one with absorption at 460 nm, showing a higher fluorescence resonance transfer efficiency than the other absorption at 395 nm. We determined the apparent Stern–Volmer quenching constant (K app) of GFP for GO to be 2.6 × 10−7 mL/g. However, GFP fluorescence disappeared upon covalent immobilization on GO.

The extremely broad odorant response profile of mouse olfactory sensory neurons expressing the odorant receptor MOR256-17 includes trace amine-associated receptor ligands.

The mouse olfactory system employs ~1100 G‐protein‐coupled odorant receptors (ORs). Each mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and the expressed OR determines the odorant response properties of the OSN. The broadest odorant response profile thus far demonstrated in native mouse OSNs is for OSNs that express the OR gene SR1 (also known as Olfr124 and MOR256‐3). Here we showed that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256‐17 (also known as Olfr15 and OR3) is even broader than that of OSNs expressing SR1. We investigated the electrophysiological properties of green fluorescent protein (GFP)+ OSNs in a MOR256‐17‐IRES‐tauGFP gene‐targeted mouse strain, in parallel with GFP+ OSNs in the SR1‐IRES‐tauGFP gene‐targeted mouse strain that we previously reported. Of 35 single chemical compounds belonging to distinct structural classes, MOR256‐17+ OSNs responded to 31 chemicals, compared with 10 for SR1+ OSNs. The 10 compounds that activated SR1+ OSNs also activated MOR256‐17+ OSNs. Interestingly, MOR256‐17+ OSNs were activated by three amines (cyclohexylamine, isopenthylamine, and phenylethylamine) that are typically viewed as ligands for chemosensory neurons in the main olfactory epithelium that express trace amine‐associated receptor genes, a family of 15 genes encoding G‐protein‐coupled receptors unrelated in sequence to ORs. We did not observe differences in membrane properties, indicating that the differences in odorant response profiles between the two OSN populations were due to the expressed OR. MOR256‐17+ OSNs appear to be at one extreme of odorant responsiveness among populations of OSNs expressing distinct OR genes in the mouse.

Ground-state proton transfer kinetics in green fluorescent protein.

Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional 13C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a 13C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site, likely the terminal proton acceptor of ESPT, Glu222. Additionally, experiments on a selection of GFP circular permutants suggest an important role played by the structural dynamics of the seventh β-strand in gating proton transfer from bulk solution to the buried chromophore.

Illuminating the origins of spectral properties of green fluorescent proteins via proteochemometric and molecular modeling.

Green fluorescent protein (GFP) has immense utility in biomedical imaging owing to its autofluorescent nature. In efforts to broaden the spectral diversity of GFP, there have been several reports of engineered mutants via rational design and random mutagenesis. Understanding the origins of spectral properties of GFP could be achieved by means of investigating its structure-activity relationship. The first quantitative structure-property relationship study for modeling the spectral properties, particularly the excitation and emission maximas, of GFP was previously proposed by us some years ago in which quantum chemical descriptors were used for model development. However, such simplified model does not consider possible effects that neighboring amino acids have on the conjugated π-system of GFP chromophore. This study describes the development of a unified proteochemometric model in which the GFP chromophore and amino acids in its vicinity are both considered in the same model. The predictive performance of the model was verified by internal and external validation as well as Y-scrambling. Our strategy provides a general solution for elucidating the contribution that specific ligand and protein descriptors have on the investigated spectral property, which may be useful in engineering novel GFP variants with desired characteristics.

Different Photochemical Events of a Genetically Encoded Phenyl Azide Define and Modulate GFP Fluorescence

Expanding the genetic code opens new avenues to modulate protein function in real time. By genetically incorporating photoreactive phenyl azide, the fluorescent properties of green fluorescent protein (GFP) can be modulated by light. Depending on the residue in GFP programmed to incorporate the phenyl azide, different effects on function and photochemical pathways are observed.

Quantitative structure–property relationship study of spectral properties of green fluorescent protein with support vector machine

Green fluorescent protein (GFP) is an autofluorescent protein that has been widely used in the biomedical sciences for molecular imaging applications. Computational approach for predicting the spectral properties of GFP offers great benefit for the design and engineering of novel color variants. Herein, we present a quantitative structure–property relationship (QSPR) study to model the spectral properties (e.g. excitation and emission maxima) of GFP chromophores using support vector machine (SVM). The data set is composed of 19 chromophores from GFP color variants and 29 synthetic GFP chromophores based on the imidazolinone scaffold. Quantum chemical descriptors were used to provide information on the physicochemical properties of the chromophores. Such descriptors were mapped onto a higher dimensional space via kernel functions (e.g. linear, polynomial and radial basis function kernels) and learning is then performed using SVM. The predicted spectral properties were well correlated with their experimental values as observed from correlation coefficient in the range of r=0.953–0.979. Predictive performance of excitation maxima (r=0.967–0.979) outperformed that of the emission maxima (r=0.953–0.961). The present strategy holds great promise for expanding the spectral repertoire of GFP by facilitating the rational design of novel color variants.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

GFP tagging sheds light on protein translocation: implications for key methods in cell biology.

Green fluorescent protein (GFP) is a powerful tool for studying gene expression, protein localization, protein-protein interactions, calcium concentrations, and redox potentials owing to its intrinsic fluorescence. However, GFP not only contains a chromophore but is also tightly folded in a temperature-dependent manner. The latter property of GFP has recently been exploited (1) to characterize the translocase of the outer mitochondrial membrane and (2) to discriminate between protein transport across and into biomembranes in vivo. I therefore suggest that GFP could be a valuable tool for the general analysis of protein transport machineries and pathways in a variety of organisms. Moreover, results from such studies could be important for the interpretation and optimization of classical experiments using GFP tagging.

Investigating structure and fluorescence properties of green fluorescent protein released from chitosan nanoparticles

Purified green fluorescent protein (GFP) was loaded into the chitosan nanoparticles to develop a model system for studying protein structure and fluorescence properties after entrapment and subsequent release. In vitro release studies of fluorescence showed that, 88% of loaded GFP was released from chitosan nanoparticles within 48h. The released GFP structure was assessed by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. The results confirmed that the GFP preserved its intrinsic conformation and fluorescence properties during entrapment process and after release. Our essential findings on structural stability of GFP in chitosan nanoparticles implied a strong prospective of chitosan nanoparticles for therapeutic protein delivery.

Quantum Chemistry Behind Bioimaging: Insights from Ab Initio Studies of Fluorescent Proteins and Their Chromophores

The unique properties of green fluorescent protein (GFP) have been harnessed in a variety of bioimaging techniques, revolutionizing many areas of the life sciences. Molecular-level understanding of the underlying photophysics provides an advantage in the design of new fluorescent proteins (FPs) with improved properties; however, because of its complexity, many aspects of the GFP photocycle remain unknown. In this Account, we discuss computational studies of FPs and their chromophores that provide qualitative insights into mechanistic details of their photocycle and the structural basis for their optical properties. In a reductionist framework, studies of well-defined model systems (such as isolated chromophores) help to understand their intrinsic properties, while calculations including protein matrix and/or solvent demonstrate, on the atomic level, how these properties are modulated by the environment. An interesting feature of several anionic FP chromophores in the gas phase is their low electron detachment energy. For example, the bright excited ππ* state of the model GFP chromophore (2.6 eV) lies above the electron detachment continuum (2.5 eV). Thus, the excited state is metastable with respect to electron detachment. This autoionizing character needs to be taken into account in interpreting gas-phase measurements and is very difficult to describe computationally. Solvation (and even microsolvation by a single water molecule) stabilizes the anionic states enough such that the resonance excited state becomes bound. However, even in stabilizing environments (such as protein or solution), the anionic chromophores have relatively low oxidation potentials and can act as light-induced electron donors. Protein appears to affect excitation energies very little (<0.1 eV), but alters ionization or electron detachment energies by several electron volts. Solvents (especially polar ones) have a pronounced effect on the chromophore's electronic states; for example, the absorption wavelength changes considerably, the ground-state barrier for cis-trans isomerization is reduced, and fluorescence quantum yield drops dramatically. Calculations reveal that these effects can be explained in terms of electrostatic interactions and polarization, as well as specific interactions such as hydrogen bonding. The availability of efficient computer implementations of predictive electronic structure methods is essential. Important challenges include developing faster codes (to enable better equilibrium sampling and excited-state dynamics modeling), creating algorithms for properties calculations (such as nonlinear optical properties), extending standard excited-state methods to autoionizing (resonance) states, and developing accurate QM/MM schemes. The results of sophisticated first-principle calculations can be interpreted in terms of simpler, qualitative molecular orbital models to explain general trends. In particular, an essential feature of the anionic GFP chromophore is an almost perfect resonance (mesomeric) interaction between two Lewis structures, giving rise to charge delocalization, bond-order scrambling, and, most importantly, allylic frontier molecular orbitals spanning the methine bridge. We demonstrate that a three-center Hückel-like model provides a useful framework for understanding properties of FPs. It can explain changes in absorption wavelength upon protonation or other structural modifications of the chromophore, the magnitude of transition dipole moment, barriers to isomerization, and even non-Condon effects in one- and two-photon absorption.

Second-Harmonic Generation in GFP-like Proteins

The second-order nonlinear optical properties of green fluorescent proteins (GFPs), such as the photoswitchable Dronpa and enhanced GFP (EGFP), have been studied at both the theoretical and experimental levels. In the case of Dronpa, both approaches are consistent in showing the rather counterintuitive result of a larger second-order nonlinear polarizability (or first hyperpolarizability, beta) for the protonated state, which has a higher transition energy, than for the deprotonated, fluorescent state with its absorption at lower energy. Moreover, the value of beta for the protonated form of Dronpa is among the highest reported for proteins. In addition to the pH dependence, we have found a wavelength dependence in the beta values. These properties are essential for the practical use of Dronpa or other GFP-like fluorescent proteins as second-order nonlinear fluorophores for symmetry-sensitive nonlinear microscopy imaging and as nonlinear optical sensors for electrophysiological processes. An accurate value of the first hyperpolarizability is also essential for any qualitative analysis of the nonlinear images.