Chromophore-protein Interactions Research Articles

Protein-Chromophore Interactions in Green Fluorescent Protein (GFP) Studied by Split Protein Reconstitution

We have utilized a split green fluorescent protein (GFP) system to generate a range of semi-synthetic proteins to study the interaction of the autocatalytically-formed chromophore with the protein environment. Using circularly permuted GFP, the alpha helix containing the chromophore has been relocated to the N-terminus and then removed by site-specific proteolysis of an engineered loop region. Following denaturation and size exclusion chromatography purification, the truncated protein and the chromophore-bearing peptide are separated. We previously showed that the truncated protein is capable of re-forming a chromophore upon reconstitution with a synthetic peptide. This work is extended to incorporate unnatural amino acids into the chromophore for studying hydrogen bonding and proton transfer. An additional application we have pursued is to use chromophore "transplantation" in order to examine the spectroscopic effects of mutations that would ordinarily be impossible due to the preclusion of chromophore formation. Arg96, in particular, is an interesting residue because of its major role in defining the electrostatic environment, however, its intolerance to mutation necessitates the use of a split protein and complementation with a chromophore-containing peptide harvested from a non-mutated donor. We use this scheme to investigate the effects that remodeling the electrostatics at this critical position have on the color tuning and quantum yield of GFP.

Temperature Dependence of the Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Protein Mplum

We used molecular dynamics (MD) simulations to investigate the dynamics of the hydrogen bonds formed due to protein-chromophore interactions in the far-red fluorescent protein mPlum. Low temperature experiments on mPlum show a reduced Stokes shift compared to room temperature. This suggests that the increased flexibility of the chromophore environment at higher temperatures along with its ability to reorganize after excitation is related to the larger Stokes shift. We performed MD simulations at various temperatures and systematically explored the protein-chromophore hydrogen bond pattern at various temperatures. We also investigated the dynamics of the hydrogen bond formed between residues E16 and I65, which is considered to play an important role in the observed large red shift in the emission spectrum. We quantify the hydrogen bonding pattern as a function of temperature to show how the Stokes shift in mPlum is correlated to the E16-I65 hydrogen bond dynamics.

Extended Stokes Shift in Fluorescent Proteins: Chromophore–Protein Interactions in a Near-Infrared TagRFP675 Variant

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10–45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

Light-dark Adaptation of Channelrhodopsin C128T Mutant

Channelrhodopsins are microbial type rhodopsins that operate as light-gated ion channels. Largely prolonged lifetimes of the conducting state of channelrhodopsin-2 may be achieved by mutations of crucial single amino acids, i.e. cysteine 128. Such mutants are of great scientific interest in the field of neurophysiology because they allow neurons to be switched on and off on demand (step function rhodopsins). Due to their slow photocycle, structural alterations of these proteins can be studied by vibrational spectroscopy in more detail than possible with wild type. Here, we present spectroscopic evidence that the photocycle of the C128T mutant involves three different dark-adapted states that are populated according to the wavelength and duration of the preceding illumination. Our results suggest an important role of multiphoton reactions and the previously described side reaction for dark state regeneration. Structural changes that cause formation and depletion of the assumed ion conducting state P520 are only small and follow larger changes that occur early and late in the photocycle, respectively. They require only minor structural rearrangements of amino acids near the retinal binding pocket and are triggered by all-trans/13-cis retinal isomerization, although additional isomerizations are also involved in the photocycle. We will discuss an extended photocycle model of this mutant on the basis of spectroscopic and electrophysiological data.

Solid‐State NMR Spectroscopy to Probe Photoactivation in Canonical Phytochromes

The photoreceptor phytochrome switches photochromically between two thermally stable states called Pr and Pfr. Here, we summarize recent solid-state magic-angle spinning (MAS) NMR work on this conversion process and interpret the functional mechanism in terms of a nano-machine. The process is initiated by a double-bond photoisomerization of the open-chain tetrapyrrole chromophore at the methine bridge connecting pyrrole rings C and D. The Pr-state chromophore and its surrounding pocket in canonical cyanobacterial and plant phytochromes has significantly less order, tends to form isoforms and is soft. Conversely, Pfr shows significantly harder chromophore-protein interactions, a well-defined protonic and charge distribution with a clear classical counterion for the positively charged tetrapyrrole system. The soft-to-hard/disorder-to-order transition involves the chromophore and its protein surroundings within a sphere of at least 5.5 Å. The relevance of this collective event for signaling is discussed. Measurement of the intermediates during the Pfr → Pr back-reaction provides insight into the well-adjusted mechanics of a two-step transformation. As both Pr → Pfr and Pfr → Pr reaction pathways are different in ground and excited states, a photochemically controlled hyper-landscape is proposed allowing for ratchet-type reaction dynamics regulating signaling activity.

Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism

Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF domain in its red-absorbing 15Z state (Pr) and the TePixJ GAF domain in its green-absorbing 15E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: (i) the chromophore of AnPixJ Pr is phycocyanobilin in a C5-Z,syn/C10-Z,syn/C15-Z,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10-Z,syn/C15-E,anti configuration, (ii) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, (iii) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15-Z and C15-E are discussed based on the X-ray structures at 1.8 and 2.0-Å resolution, respectively, in two distinct configurations.

Spectral "Fine" Tuning in Fluorescent Proteins: The Case of the GFP-Like Chromophore in the Anionic Protonation State.

Fluorescent proteins (FPs), featuring the same chromophore but different chromophore-protein 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 using 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 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 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.

Molecular-Level Insight into the Spectral Tuning Mechanism of the DsRed Chromophore.

We present a detailed study of the protein environmental effects on the one- and two-photon absorption (1PA and 2PA, respectively) properties of the S0-S1 transition in the DsRed protein using the polarizable embedding density functional theory formalism. We find that steric factors and chromophore-protein interactions act in concert to enhance the 2PA activity inside the protein while adversely blue-shifting the 1PA maximum. A two-state model reveals that the 2PA intensity gain is primarily governed by the increased change in the permanent dipole moment between the ground and the excited states acquired inside the protein. Our results indicate that this mainly is attributable to counter-directional contributions stemming from Lys163 and the conserved Arg95 with the former additionally identified as a key residue in the color tuning mechanism. The results provide new insight into the tuning mechanism of DsRed and suggest a possible strategy for simultaneous improvement of its 1PA and 2PA properties.

Model of Abnormal Chromophore-Protein Interaction for Е181К Rhodopsin Mutation: Computer Molecular Dynamics Study.

The interaction of the 11-cis-retinal chromophore with the surrounding amino acid residues in the chromophore center of the rhodopsin protein has been investigated for the Е181К mutant form using molecular dynamics simulation. A comparative analysis of the arrangement of the amino acid residues in the chromophore center has been performed for both wild (native) and mutant rhodopsins. It is shown that for the Е181К mutant rhodopsin there is no proper binding of 11-cis-retinal with the surrounding amino acid residues. The distortion of the conformation states in the mutant rhodopsin molecule takes place in both the chromophore center and cytoplasmic domain. Our simulations suggest that a stable covalent linkage of 11-cis-retinal with the protein part (viz. opsin) of the rhodopsin molecule will not form. This, on the other hand, implies that the protein’s active site in the cytoplasmic domain, which is responsible for the G-protein binding (so-called transducin), may not be completely blocked.Based on our molecular simulation data, we discuss the possible correlation between retinitis pigmentosa pathogenesis and the structural and functional properties of the rhodopsin protein.

The influence of chromophore-protein interactions on spectroscopic properties of the yellow fluorescent protein

The influence of chromophore-protein interactions on spectroscopic properties of the yellow fluorescent protein

Solid-State NMR Spectroscopic Study of Chromophore–Protein Interactions in the Pr Ground State of Plant Phytochrome A

Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1H-15N experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.

Atomistic Study of the Long-Lived Quantum Coherences in the Fenna-Matthews-Olson Complex

A remarkable amount of theoretical research has been carried out to elucidate the physical origins of the recently observed long-lived quantum coherence in the electronic energy transfer process in biological photosynthetic systems. Although successful in many respects, several widely used descriptions only include an effective treatment of the protein-chromophore interactions. In this work, by combining an all-atom molecular dynamics simulation, time-dependent density functional theory, and open quantum system approaches, we successfully simulate the dynamics of the electronic energy transfer of the Fenna-Matthews-Olson pigment-protein complex. The resulting characteristic beating of populations and quantum coherences is in good agreement with the experimental results and the hierarchy equation of motion approach. The experimental absorption, linear, and circular dichroism spectra and dephasing rates are recovered at two different temperatures. In addition, we provide an extension of our method to include zero-point fluctuations of the vibrational environment. This work thus presents, to our knowledge, one of the first steps to explain the role of excitonic quantum coherence in photosynthetic light-harvesting complexes based on their atomistic and molecular description.

Exploring Chromophore-Binding Pocket: High-Resolution Solid-State 1H–13C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme

High-resolution two-dimensional (2D) 1H–13C heteronuclear correlation spectra are recorded for selective observation of interfacial 3–5.5 Å contacts of the uniformly 13C-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI–HETCOR) method. For improving 1H spectral resolution, a windowed phase-modulated Lee–Goldburg (wPMLG) decoupling scheme is applied during the t 1 evolution period. Our approach allows for identification of chromophore–protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1–514, Cph1Δ2) containing uniformly 13C- and 15N-labeled PCB chromophore (u-[13C,15N]-PCB-Cph1Δ2) at 17.6 T.

Molecular Science of Rhodopsins

Rhodopsins contain a retinal molecule, and convert light into chemical energy or signal. The chromophore of visual or microbial rhodopsins is a retinal Schiff base of the 11-cis or all-trans form, respectively, where specific chromophore-protein interaction determines their colors. Upon light absorption, ultrafast photoisomerization initiates protein structural changes, leading to each functional expression. By use of spectroscopic methods, we have been studying how rhodopsins respond to light. Ultrafast spectroscopy of visual rhodopsin revealed that cis-trans isomerization is the primary event in our vision, which is optimized in protein environment. Fourier-transform infrared (FTIR) spectroscopy of visual and microbial rhodopsins provides various important vibrational bands related to structural changes of these proteins. Detection of protein-bound water molecules is one of the research highlights, and the comprehensive FTIR study has shown that a strongly hydrogen-bonded water molecule is the functional determinant of light-driven proton pump proteins. Here I review our spectroscopic challenge for > 25 years, particularly focusing our recent findings.

Modulating Native-like Residual Structure in the Fully Denatured State of Photoactive Yellow Protein Affects Its Refolding

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (DeltaC(p)), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is approximately 20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.

Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface

Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S(0) and excited state S(1) are modified by the protein. It is widely assumed that changes in energy gap between S(0) and S(1) are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S(1) energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S(1) energy surface.

The Role of the Chromophore in the Biological Photoreceptor Phytochrome: An Approach Using Chemically Synthesized Tetrapyrroles

In plants and bacteria, phytochromes serve as light-inducible, red-/far-red light sensitive photoreceptors that control a wide range of photomorphogenetic processes. Phytochromes comprise a protein moiety and a covalently bound bilin chromophore. Bilins are open-chain tetrapyrrole compounds that derive biosynthetically from ubiquitous porphyrins. The investigations of phytochromes reveal that precise interactions between the protein moiety and its bilin chromophore are essential for the proper functioning of this photoreceptor; accordingly, synthetic manipulation of the parts is an important method for studying the whole. Although variations in the protein structure are readily accomplished by routine mutagenesis protocols, the generation of structurally modified bilins is a laborious, multistep process. Recent improvement in the synthesis of open-chain tetrapyrroles now permits the generation of novel, structurally modified (and even selectively isotope-labeled) chromophores. Furthermore, by using the capability of recombinant apo-phytochrome to bind the chromophore autocatalytically, researchers can now generate novel chromoproteins with modified functions. In the protein-bound state, the phytochrome chromophore is photoisomerized at one double bond, in the bridge between the last two of the four pyrrole rings (the C and D rings), generating the thermally stable, physiologically active P(fr) form. This conversion--photoisomerization from the form absorbing red light (P(r)) to the form absorbing far-red light (P(fr))--covers 12 orders of magnitude, from subpicoseconds to seconds. Such spectroscopic and kinetic studies yield a wealth of time-resolved spectral data, even more so, if proteins with changed sequence or chromophore structure are utilized. In particular, bilins with a changed substitution pattern at the photoisomerizing ring D have shed light on the chromophore-protein interactions during the photoisomerization. The mechanisms generating and stabilizing the light-induced P(fr) form of phytochromes are now seen in greater detail. On the other hand, the use of bilins with selective incorporation of stable isotopes identify light-induced conformational motions when studied by vibrational (FTIR and Raman) and NMR spectroscopy. In this Account, we present spectroscopic investigations that provide structural details in these biological photoreceptors with great precision and document the dynamics elicited by light excitation. This approach yields important information that complements the data deduced from crystal structure.

Chromophore Structure of Cyanobacterial Phytochrome Cph1 in the Pr State: Reconciling Structural and Spectroscopic Data by QM/MM Calculations

A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His260 in the chromophore-binding pocket such that either the δ-nitrogen (M-HSD) or the ɛ-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His260 with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.

The Photoreactions of Recombinant Phytochrome CphA from the Cyanobacterium Calothrix PCC7601: A Low‐Temperature UV–Vis and FTIR Study

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV-Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV-Vis and FTIR experiments. Three intermediates have been trapped at -25, -45 and -120 degrees C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at -70 and -140 degrees C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore-protein interactions.

Glutamic Acid 181 Is Uncharged in Dark-Adapted Visual Rhodopsin

We have performed a high-level quantum chemical analysis to study the chromophore-protein interaction involving the charged (B) and/or uncharged (C) form of E181 and also a mutant E181Q model in the presence of the primary counterion E113 (A). As the magnitude of the calculated spectral shifts on either side remains within +/-10 nm, we show that the orientation of the dipole moment vector is the key to unlocking the puzzle on this contentious issue. We find that E181 is present in the uncharged (or) protonated form in the dark-adapted visual Rhodopsin, and therefore an electrostatically neutral environment is envisaged.