First-Principles Characterization of the Energy Landscape and Optical Spectra of Green Fluorescent Protein along the A→I→B Proton Transfer Route

Grigorenko, Bella L.; Nemukhin, Alexander V.; Polyakov, Igor V.; Morozov, Dmitry; Krylov, Anna I.

doi:10.1021/ja402472y

Cited by 68 publications

(139 citation statements)

References 56 publications

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“…This method was successfully employed in numerous applications, including simulations of the c‐GMP hydrolysis in water, studies of proton transfer routes in the photocycle of the green fluorescent protein, modeling chemical transformations upon penicillin G hydrolysis by penicillin acylase . In every case a reaction mechanism was revealed by computing a set of stationary points on the potential energy surface corresponding to reaction intermediates and transition states.…”

Section: Models and Methodsmentioning

confidence: 99%

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

2016

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We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc.

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Section: Models and Methodsmentioning

confidence: 99%

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

2016

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“…40 This assignment was motivated by the small blue shift of 0.02−0.04 (depending on the quantum method) computed between the excitation energies of their B and I forms and the fact that, experimentally, the 0 →0 transition of the B form is also blue-shifted (albeit by 0.1 eV) with respect to the I form.…”

Section: Alternative H-bond Network For the B Formmentioning

confidence: 99%

Chromophore–Protein Coupling beyond Nonpolarizable Models: Understanding Absorption in Green Fluorescent Protein

Daday

Curutchet

Sinicropi

et al. 2015

J. Chem. Theory Comput.

122

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The nature of the coupling of the photoexcited chromophore with the environment in a prototypical system like green fluorescent protein (GFP) is to date not understood, and its description still defies state-of-the-art multiscale approaches. To identify which theoretical framework of the chromophore-protein complex can realistically capture its essence, we employ here a variety of electronic-structure methods, namely, time-dependent density functional theory (TD-DFT), multireference perturbation theory (NEVPT2 and CASPT2), and quantum Monte Carlo (QMC) in combination with static point charges (QM/MM), DFT embedding (QM/DFT), and classical polarizable embedding through induced dipoles (QM/MMpol). Since structural modifications can significantly affect the photophysics of GFP, we also account for thermal fluctuations through extensive molecular dynamics simulations. We find that a treatment of the protein through static point charges leads to significantly blue-shifted excitation energies and that including thermal fluctuations does not cure the coarseness of the MM description. While TDDFT calculations on large cluster models indicate the need of a responsive protein, this response is not simply electrostatic: An improved description of the protein in the ground state or in response to the excitation of the chromophore via ground-state or state-specific DFT and MMpol embedding does not significantly modify the results obtained with static point charges. Through the use of QM/MMpol in a linear response formulation, a different picture in fact emerges in which the main environment response to the chromophore excitation is the one coupling the transition density and the corresponding induced dipoles. Such interaction leads to significant red-shifts and a satisfactory agreement with full QM cluster calculations at the same level of theory. Our findings demonstrate that, ultimately, faithfully capturing the effects of the environment in GFP requires a quantum treatment of large photoexcited regions but that a QM/classical model can be a useful approximation when extended beyond the electrostatic-only formulation.

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“…In contrast, spectroscopy on wildtype (wt)GFP included infrared pump probe (16), time-resolved fluorescence (17)(18)(19), transient infrared (20,21), and femtosecond Raman spectroscopy (22), as well as computational studies (23)(24)(25), providing a fairly complete picture of the photophysical and photochemical steps leading to green fluorescence (26,27). In the electronic ground state (GS), wtGFP exists as a mixture of neutral chromophore (A, ∼400 nm peak absorbance) and a small population of anionic chromophore (B, ∼475 nm peak Significance Fluorescent proteins (FPs) started their incredible, colorful journey in bioimaging and biomedicine with the extraction and purification of GFP from the Pacific jellyfish Aequorea victoria more than 50 years ago.…”

mentioning

confidence: 99%

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

Oscar

Liu

Zhao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

191

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Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca 2+ ) sensing. This study reveals that, in the absence of Ca 2+ , the dominant skeletal motion is a ∼170 cm −1 phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca 2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca 2+ in physiologically relevant environments.calcium-sensing fluorescent protein | femtosecond Raman spectroscopy | fluorescence modulation mechanism | molecular movie G reen fluorescent protein (GFP) first emerged as a revolutionary tool for bioimaging and molecular and cellular biology about 20 years ago (1-3), and the quest to discover and engineer biosensors with improved and expanded functionality has yielded exciting advances. Recently, the color palette of genetically encoded Ca 2+ sensors for optical imaging (the GECO series) has been expanded to include blue, improved green, red intensiometric, and emission ratiometric sensors (4-7). The GECO proteins belong to the GCaMP family of Ca 2+ sensors that are chimeras of a circularly permutated (cp)GFP, calmodulin (CaM), and a peptide derived from myosin light chain kinase (M13) (8). The CaM unit undergoes large-scale structural changes upon Ca 2+ binding as it wraps around M13. These changes, especially at the interfacial region where CaM interacts with cpGFP, allosterically alter the local environment of the tyrosine-derived chromophore and lead to dramatic fluorescence change in the presence of Ca 2+ (9, 10). Because GCaMP and GECO proteins are genetically encodable, show sensitivity to physiologically relevant Ca 2+ concentrations, and respond to Ca 2+ concentration changes rapidly, they have gained increasing popularity for in vivo imaging of Ca 2+ in neur...

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First-Principles Characterization of the Energy Landscape and Optical Spectra of Green Fluorescent Protein along the A→I→B Proton Transfer Route

Cited by 68 publications

References 56 publications

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

Chromophore–Protein Coupling beyond Nonpolarizable Models: Understanding Absorption in Green Fluorescent Protein

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

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