Excited State Electronic Interconversion and Structural Transformation

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Excited State Electronic Interconversion and Structural Transformation of Engineered Red Emitting Green Fluorescent Protein Mutant George Augustine, sriram Srinivasa raghavan, Kamini NumbiRamudu, Shanmugam Easwaramoorthi, Ganesh Shanmugam, Jaimohan Seetharani Murugaiyan, Krishnasamy Gunasekaran, Chinju Govind, Venugopal Karunakaran, and Niraikulam Ayyadurai J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b10516 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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The Journal of Physical Chemistry

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Excited State Electronic Interconversion and Structural Transformation of

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Engineered Red Emitting Green Fluorescent Protein Mutant

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George Augustine ‡* Sriram Raghavan, †* Kamini NumbiRamudu, ‡ Shanmugam Easwaramoorthi, § Ganesh

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Shanmugam,⊥ Jaimohan Seetharani Murugaiyan║ Krishnasamy Gunasekaran,† Chinju Govind∇ Venugopal

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Karunakaran, ∇ and Niraikulam Ayyadurai, ‡

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AUTHOR ADDRESS

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‡Department

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Leather Research Institute (CSIR-CLRI), Chennai, India, 600 020.

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† Department

of Biochemistry and Biotechnology, Council of Scientific and Industrial Research-Central

of Crystallography and Biophysics, University of Madras, Chennai, India, 600 025.

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§ Inorganic

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⊥ Organic

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║ Division

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∇

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National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695 019,

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Kerala, India

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AUTHOR INFORMATION

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Corresponding Author

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‡ Dr.

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Chennai, India. [email protected]

& Physical Chemistry Laboratory, CSIR-CLRI, India, 600 020.

& Bio-organic Chemistry, CSIR-CLRI, India, 600 020. of Advanced Materials, CSIR-CLRI, Chennai, India, 600 020.

Photosciences and Photonics Section, Chemical Sciences and Technology Division, CSIR-

N. Ayyadurai, Division of Biochemistry and Biotechnology, CSIR-CLRI, Adyar,

ACS Paragon Plus Environment

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Red-emitting fluorescent protein (RFP) with large Stokes shift offer limited auto-fluorescence background

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used in deep tissue imaging. Here, by introducing the free amino group in Aequorea victoria, the

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electrostatic charges of p-hydroxybenzylidene imidazolinone (p-HBI) chromophore of green fluorescent

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protein (GFP) has been altered resulting in an unusual, 85 nm red shifted fluorescence. The structural and

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biophysical analysis suggested that the red shift is due to positional shift occupancy of Glu222 and Arg96

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resulted along with extended conjugation and a relaxed chromophore. Femtosecond transient absorption

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spectra exhibited that the excited state relaxation dynamics of rGFP (τ4 = 234 ps) are faster compared to the

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avGFP (τ4 = 3.0 ns). The nanosecond time-resolved emission spectra of rGFP reveal the continuous spectral

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shift during emission by a solvent reorientation in the chromophore. Finally, the molecular dynamics

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simulations revealed the rearrangement of the hydrogen bond interactions in the chromophore vicinity,

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reshaped the symmetric distribution of Van der Waal space to fine tune the GFP structure resulting with

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highly red shifted rGFP.

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1. Introduction

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Aequorea victoria green fluorescent proteins (avGFP) have remarkably revolutionized the area of cell

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biology research attributed to the inherent fluorescence quantum yields.1-4 Later, the red fluorescent proteins

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(RFP) have encountered considerable attention than GFPs, as they have enhanced fluorescence emission

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above 600 nm, which is more advantageous in bio-imaging owing to their reduced autofluoresence, lower

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light scattering, and higher tissue penetration.5 In general, the RFP variants share similar chromophore core

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motif of GFPs namely p-hydroxybenzylidene-imidazolinone (p-HBI) except for the additional acylimine

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(C=N-C=O) group at the C1 atom of the Cα-Cβ bond of tyrosine (Tyr66), which is formed through

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oxidation and have an extended π-conjugated network.6 This extended conjugation network induces

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additional resonance states thereby decreasing the electronic energy gap leading to red shifted fluorescence.

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Several research groups have aimed to mimic the structure in avGFP with red-shifted excitation/emission

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spectra either through functional group modification of the chromophore or by changing the protein

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environment perturbing specific interactions. For instance, Yellow fluorescent protein (YFP) (Thr203)

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evolved from avGFP, forms π-stacking interactions with an p-HBI chromophore ensuing in a notable red-

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shifted spectrum (20 nm).7 Moreover, Mishin research group attempted in creating red chromophore in

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avGFP through the non-oxidative mechanism of Ser65 dehydration resulting in a double bond formation

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between Cα and Cβ atoms of Asn68 and Glu222, respectively.8 Likewise, Budisa and colleagues introduced

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the tryptophan analogue in the cyan fluorescent protein leading to a golden variant with a large Stokes

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shift.9 Primarily, RFP was originated from Anthozoa species10 after which several red emitting RFPs (620

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nm) were constructed by the combination of rational design and stepwise random mutagenesis, directed

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evolution and somatic hyper mutation.11-12 Though GFP and RFP share 99% sequence similarity, the

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folding ability and spectroscopical properties of both proteins are different. However, it is still challenging

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to develop a RFP with large Stokes shift values by mimicking avGFP variant. Here, we hypothesized that

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the reorganization of cis-trans isomerization of chromophore Tyr66 of the non-intrinsic photoswitch

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forming GFP-HS variant which may result with the large red shift. For that, we genetically introduced the


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amine chemistry in the ortho-position of Tyr66 to mimic the electrostatic charge similar to RFP. As

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expected, the site-specific and global incorporation of 3-amino-L-tyrosine (3-NH2-L-Tyr) in the avGFP

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resulted in a mimic of RFP (rGFP) chromophore containing protein. Herein, we report that the positional

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reorientation of the chromophore by introducing additional amine interacting moiety (Tyr66) in GFP which

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yielded 85 nm red-shifted green fluorescent protein (rGFP) compared with the wild counterpart. The amino

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substitution also has effects on physiological properties such as oligomeric state, excited state energy, pH

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response and etc. Comparative investigations of FP’s were also evaluated through small angle X-ray

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scattering (SAXs), steady-state and time-resolved fluorescence studies. Femtosecond time-resolved

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transient absorption spectra and molecular dynamics simulation were carried out to identify the altered

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excited state relaxation path and isomeric effects (cis-trans) on rGFP. The effect of water hydration channel

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and solvent occupiable spheres were studied to understand the altered space in avGFP and rGFP,

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respectively. Geometrical contact alteration upon addition of NH2 group in the chromophore was compared

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with GFP, which was studied using structural, biophysical and molecular dynamics simulations. Finally,

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nanosecond time-resolved emission spectral (TRES) studies of rGFP and its native GFP reveals the lacking

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of critical inter-molecular interaction with the chromophore.

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2. Methods

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2.1 Site Specific incorporation of Non-canonical amino acids

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Site specific incorporation of 3-NH2-L-Tyr was performed in E. coli JW2581 containing pAD-GFPY66am

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and pAC-DOPA- 6TRN (non-canonical tRNA/synthetase) as mentioned in Ayyadurai et al.13, 14 (2011) The

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cells having both the plasmids for site specific incorporation were grown in LB broth supplemented with

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ampicillin (50 mg/mL) and tetracycline (7.5 mg/mL) then was incubated overnight at 37 °C. The cells in

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the overnight cultures were collected, washed thoroughly in phosphate buffer saline (PBS), and inoculated

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into glycerol minimal media (MM) supplemented with 0.03 mM tyrosine (Tyr), ampicillin (50 mg/mL) and

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tetracycline (7.5 mg/mL). The cultures were allowed to grow at 37°C until attaining the mid log phase (A600


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0.6 to 0.8) and 1 mM of 3-NH2-L-Tyr was added before induction. The optimum expression of site

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specifically incorporated 3-NH2-L-Tyr was achieved upon 0.2% arabinose induction for 7 h at 37 °C.

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Interestingly, rGFP was obtained from both site-specific and global incorporation methods. Then, the cells

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were harvested and frozen at – 80 °C for purification.

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2.2 Protein expression and Purification

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The pET28-avGFP plasmid containing the avGFP gene was successfully cloned into pQE80L using

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BamH1 and HindIII restriction enzyme, as described by George et al. (2017).15 The avGFP

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expression was induced in Escherichia coli tyrosine auxotroph JW2581 by the addition of 1 mM

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IPTG in Luria Bertani (LB) broth and incubated at 37 oC for 6 h. At the same time, 3-NH2-L-Tyr

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labeled protein expression was also carried out in the same E. coli auxotroph cells harboring

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pQE80L-avGFP. The cells were first grown in minimal medium consisting of 0.3 mM tyrosine as

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a natural analogue, until the depletion of tyrosine in cells during the mid-log phase (A600 0.6 to 0.8

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OD). Consequently, the tyrosine substrate analogue 3-NH2-L-Tyr was added and induced for the

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target gene expression by adding 1 mM IPTG. The optimum level of rGFP (3-NH2-L-Tyr

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incorporated) expression was obtained under these conditions. After the expression, the cells were

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ruptured in a high-pressure homogenizer (Stansted, Essex, UK) and the chromophore-containing

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avGFP (green) and rGFP (red) proteins were purified with the Ni-NTA affinity column. Further,

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the His-tag purified protein was subjected to the Gel permeation chromatography (GPC) by AKTA

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Explorer FPLC system containing Sephadex G25 HR and Superdex 200 HR column at 37 oC.

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2.3 Crystallization, Data collection, structure solution statistics.

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The avGFP was crystallized using vapor diffusion, hanging and sitting drop method at 293 K with

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a protein concentration of about 10 mg/ml in a well buffer containing 10 % PEG 8000, 100 mM

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HEPES pH 8.0 and 100 mM MgCl2. Spiky needles like crystal grew within a day. Micro seeding


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was carried out to increase the size of the crystal and hexagonal prism type crystal morphology was

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observed. The data were collected at 100 K with double crystal single wavelength monochromatic

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beam source with an experimental wavelength of 0.98 Å. The data was collected using pixel

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detector Dectris Pilatus 2 M at Elettra’s beam line 5.2 R light source (Italy), 3.2 Å was integrated

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using XDS and consequently scaled and merged using SCALA.16-18 The initial model was accessed

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by PHENIX

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presence of non-crystallographic or crystal twinning present in the data. Initially, all anisotropic

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factors B were evaluated for occurrence of anomalies due to poor data strength and low-resolution

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completion, based on bijvoet difference. No pseudo-translation was found in the model; hence, the

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data was set to isotopic for further refinement. PHENIX phaser using maximum probability was

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used to solve the structure, with a model template of PDB ID: 3V3D used for Molecular

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replacement at 1.95 Å.19 The 3V3D model was fixed by removing all the water molecules. Initially,

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rigid body refinement procedure was utilized employing real-space refinement. The Chromophore

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was fixed and restrained with phoenix- elbow refinement. The model was validated using

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MolProbity analysis for any statistical deviance.20

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The xtriage routine was conducted to evaluate the obtained model, to know the

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3. Results and discussion

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3.1 Development of congener rGFP and its spectral characterization

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To mimic RFP, we introduced an electron donating amino group in the ortho-position of the Tyr residue of

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anionic avGFP, by employing misaminoacylation21 and orthogonal translational machinery.22 avGFP and

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congener rGFP were purified with Ni-NTA affinity column, to obtain a final concentration around ~28 and

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20 mgL-1, respectively. Furthermore, the quality of NH2-Tyr incorporation was ensured by SDS-PAGE

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(Supporting Information, Figure S1), and Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF)

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analysis. A good correlation between calculated mass (28266.7 Da) and determined mass (28266.1 Da) was


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found, which are shown in Supporting Information, Figure S2. The MALDI-TOF analysis confirmed that

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the purified GFP variants with Tyr analogues possessed an incorporation efficiency of >90%, which

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resulted in “Red” GFP (rGFP). The “rGFP” obtained from the site and residue-specific incorporation

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method exhibited absorption and emission maximum at

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respectively (Supporting Information, Figure S9A&B). The emission maxima of rGFP were observed at

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610 nm which is red-shifted to the tune of 85 nm (0.34 eV) when compared to that of avGFP (525 nm, 2.40

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eV) (Supporting Information, Figure S9A&B). Previously, several attempts have been made to create the

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most-red shifted proteins from anionic GFP chromophore. To our knowledge, the developed rGFP is the

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most red shifted variant of avGFP characterized to date apart from golden FP 9 (λabs = 466 nm, λem = 574

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nm) and EYFP23 (λabs = 514 nm, λem = 527 nm). The emission maxima of rGFP was about 27 nm (0.082

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eV) red shifted from commercially available red fluorescent protein (DsRed, λem = 583 nm). 24 It is similar

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to DsRed derived mKeima (λem = 620 nm), mKate variant (LSSmKate-2 λem = 605 nm), mcherry (λem = 611

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nm) and mRuby (λem = 605 nm) variants.25-27 The extended red shifts of DsRed derivatives are not only

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attributed to the mutations introduced in the chromophore, but also to the rearrangements in the interactions

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(hydrogen bonding, electrostatic and hydrophobic) of the chromophore with surrounding key amino acid

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residues. For example, the large Stokes shift has been explained by various mechanisms. mPlum had been

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created with extended Stokes shift due to solvent reorganization28 and excited state relaxation of the

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chromophore, enabled by 2.56 Å flexible hydrogen bond between the Ile 65 (Chromophore residue) and

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Glu16.29 On the other hand, Tag RFP675’s large Stokes shift was due to the re-establishment of dipole-

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dipole interaction between the chromophore and protein matrix.30 In case of the mKeima,25 LSS mKate,27

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mBeRFP

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(ESPT). Based on the above genetic incorporation, spectroscopic and theoretical studies, we assumed that

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the enhanced red shift caused by mutations (NH2-Tyr) could result in DsRed mimic of avGFP variant.

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3.2 X-ray crystallographic investigations and rGFP modelling.

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and mCherry,

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525 nm (2.37eV) and at

610 nm (2.03eV),

large Stokes shift were provided by engineered excited state proton transfer


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The structural basis for the extended Stokes shift, the 3D structure of avGFP were obtained using X-ray

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crystallography (Supporting Information, Table S1), which was solved at physiological pH 7.4 with a

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resolution of 3.2 Å (Protein Data Bank, PDB: 5WWK). From crystallographic structural investigation, we

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determined the hydrogen bond distance between chromophore and surrounding key residues, which were

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as follows: OG Ser205–OE1 Glu222 was 4.79 Å, OG Ser205–HOH was 2.31 Å, O Asn146–HOH was 2.77

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Å, OG Thr 203-HOH was 3 Å and O Asn 146–ND1 His148 was 4.17 Å. The hydrogen bonds generally

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have energies between 0.2–40 kcal mol-1 with a bond length ranging between 2.2–2.5 Å, which are

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considered as strong, 2.5-3.2 Å as moderate and 3.2-4.0 Å as weak bond.33 Dynamic bond positioning

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average may vary, but there was a proton contact between His148-Asn146 and Ser205-Glu222, which was

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contributing to the weak electrostatic interaction. Electron density map with 1.80-sigma cut-off of 2Fo-Fc

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form represented continuous electron density between O2 CR2 – NH1, NH2-Arg 96, which had strong

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hydrogen bond of 2.46 Å and 2.80 Å (Supporting Information Figure S6), respectively. The solvent voids

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of overall avGFP and its active site region are shown in Figure 1A (1-2). The rGFP was also diffracted to

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a resolution of 5 Å and was resilient to higher quality crystallographic trials. Hence, SAXs profiling and

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protein modeling were carried out for rGFP in comparison with avGFP. Through molecular dynamics

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approach, we computationally constructed large red shifted rGFP by adding NH2 group in the ortho position

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of Tyr residue in the GFP chromophore. For full computational details, see the supporting information. As

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a result, the 3D structure of the rGFP was modeled with chromophore consisting of o-amino p-

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hydroxybenzylidene-imidazolinone (oApHBI). To discern the structural basis for red shifted absorption

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and emission in rGFP, avGFP was superimposed with rGFP. Even though the backbone key residues Val60,

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Thr61, Gln91, and Gln219 of avGFP are superimposable with rGFP backbone, residues Arg96, ASN146

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and Glu222 were significantly deviated from their position, to accommodate the oApHBI chromophore

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within the compact β barrel (Figure 1B3). Also, the electrostatic landscape of rGFP was slightly altered in

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comparison with GFP, which is shown in Figure 1C. These residues deviation caused an alteration in the H

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bond rearrangement (Supporting Information, Figure. S20) and electrostatic interaction between the

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chromophore and key residues in rGFP (Figure 1C).


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3.3 SAX investigation

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The rGFP was resilient to crystallization and is oligomeric in nature, resembling most tetrameric red shifted

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FPs (tetrameric E2 orange and E2-Crimson).34, 35 Before the SAXs modeling, the proteins were subjected

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to dynamic light scattering (DLS) to resolve the protein state (Supporting Information, Figure. S7). The

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rGFP showed high polydispersity with a broad peak (750 nm) indicating the predominance of oligomeric

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species (Supporting Information, Figure. S7B). In contrast, smaller sizes for avGFP were obtained

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reflecting the formation of a monomeric form of the protein in the solution (Supporting Information, Figure

8

S7A). Hence, SAXs profiles were acquired to determine the dynamic behavior of the rGFP and avGFP, to

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understand their corresponding oligomeric state, monodispersity, dynamic motions (determined by the

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average radius of gyration, Rg) 36- 37 (Supporting Information, Figure S4 & Table S2). The intensity profile

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of avGFP and rGFP were plotted as double logarithmic scales (I (Q) vs. Log Q), to compare the monomeric

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to oligomeric states of the protein (Supporting Information, Figure S4.3). Guinier’s linear fit was plotted to

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understand the monodispersity of the proteins (Supporting Information, Figure. S4.1). Radius of gyration

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(Rg) and Radius of cross section (Rc) values of rGFP were determined as 34.5 nm and 4.9 nm, respectively

15

(Supporting Information, Table S2). Persistent length (L) or compactness of the scattering biomolecule was

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found to be 30 nm. Kratky plot (Fig. 4-2) [I (Q)*Q2 vs. Q] of SAXs data showed bell-shaped curves that

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exhibited prolate nature of the protein with defined particle size. Similar to Kratky plot, pair wise distance

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distribution P(r) analysis of the protein was also used to calculate Dmax value of the full-length protein using

19

indirect Fourier transformation analysis (Supporting Information, Figure S4.4). To obtain a better insight

20

of the structural nature of the protein, the monomeric GFP having p2 symmetry was fitted to dummy atom

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model of rGFP having p32 symmetry, which demonstrated oligomeric propensity along with χ2 scalar fitting

22

values with agreeable deviations (Supporting Information, Table S2). The superimposed dummy scattered

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model of rGFP and avGFP are shown in Supporting information, Figure (4.5).

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3.4 Molecular dynamic simulation and QM/MM


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To comprehend, the excited state absorption at molecular level, the chromophore of GFP and rGFP in the

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gas phase has extensively been studied using molecular dynamics simulations (QM/MM). During the

3

current study, it was found that the excited state spectra displayed a major peak at around 418 nm (2.96 eV)

4

for avGFP and red shifted band at 498 nm (2.48 eV) for rGFP. Despite variation in the computed values,

5

the absorption energy mentioned above for rGFP is red shifted by 0.48 eV (80 nm) relative to the avGFP.

6

We noticed acceptable correlations between computed excitation energies and corresponding experimental

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absorption band maxima of avGFP (2.37 eV) and rGFP (2.46 eV), within the established standard

8

deviations of QM/MM protocol. Indeed, this pronounced 0.48 eV energy difference prompted us to

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investigate the nature of the rGFP’s chromophore conformation (cis-trans) at the excited state through

10

molecular simulation. It has been reported that the spectral perturbation arising from torsional and dihedral

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angle variations around the chromophore is proposed to play a vital role in spectral deviations.38,

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Substitution of Asp148 in GFP variant (S65T/H148D) had led to an increased trans-isomer conformational

13

probability known as asynchronous hula twist, attributed to the altered excited state.40 In our case, we found

14

that cis and trans conformations of the chromophore are prevalent in wild GFP and rGFP, respectively.

15

Conformational densities of dihedral angle (ø, τ) variations obtained by MD for cis-trans isomerism

16

concerning spatial occupancy are shown in Figure 2A and Figure S11-13. From these results, we observed

17

that the ethylene linker between the imidazolone and benzyl ring of Tyr in the core chromophore did not

18

show any major distortions and are within limited standard deviations. Main spectral perturbation (extended

19

red shift) can only be attributed to the incorporation of NH2-Tyr along with acquired trans-isomers in rGFP.

20

As per the previous report, excited state dynamics of Wt GFP chromophores (p-HBI) are governed by

21

Excited State Proton Transfer (ESPT), in which proton shuttles via connected networks of His148, Asp146,

22

Thr203, Ser205, and Glu222. But for the targeted proton transfer (PT) to occur, the H-bond distance

23

between the p-hydroxyphenyl group of the chromophore (donor) and the carbonyl group of Glu (acceptor)

24

should be very short .41 In particular, the central core (p-HBI) consisting of ionizable phenoxy group is

25

directly attributed to the formation of a hydrogen bond with His148 (gatekeeper), which is responsible for

39


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proton transfer only in Wt GFP but not in GFP variants (sf GFP and EGFP).42,43 X-ray crystallography

2

studies also confirmed this hypothesis. Based on these observations, we also anticipated the related

3

mechanism of proton transfer exhibiting a red shift in rGFP in comparison with avGFP. In contrast, through

4

crystallographic study, we found that the hydrogen bond distances between the chromophore and

5

surrounding residues are long, which can be classified in the range of moderate to weak categories

6

concerning bond strengths, as described above. Interestingly, according to our GFP crystal structure, we

7

deduced that His148 in avGFP and rGFP do not display direct Hydrogen bond interaction with the phenoxy

8

group of p-HBI. In detail, upon excitation, the hydroxyl group in GFP’s aromatic chromophore gets ionized

9

and tends to lose the proton leading to the formation of the anionic state. At this stage, the anionic phenolate

10

group is stabilized (balanced) by His148 residue through the water-mediated hydrogen bond network.

11

When hydroxyl group acquires partial negativity, nitrogen in imidazolone attains partial positive charge

12

and is stabilized by Glu222 network through ESPT. The system works like an electrostatic assembly similar

13

to the one given in Figure 3D, where the entire electrostatic charge network is counter balanced.

14

Introducing additional amine group in anionic avGFP tends to form self zwitterionic assembly in its

15

chromophore shown in Figure 3C. In detail, partial charge species association on NH2 group is more

16

possible than the imine in imidazolone, which is due to the proximity of amine group to phenolate moiety

17

of chromophore. Here, the distance between the oxygen of hydroxyl and nitrogen in the amine is around

18

2.6 Å, which lies in the strong hydrogen bond region. The radial hydrogen bond distance is of 1.4 Å.

19

Incorporation of amine in ortho position not only led to the spectral change but the chromophore

20

benzylidene moiety considerably shifted its locus towards polar Thr203 and Ser205. Additional pH

21

dependent spectral studies were also carried out to confirm the charge species of rGFP. The UV-visible

22

absorption spectra of rGFP between the pH 5 and pH 7 distinguish the spectral features corresponding to

23

the deprotonated and zwitterionic form (Supporting information, Figure S9E). At pH 5, the absorption

24

observed with the maximum at ~400 nm is can be attributed to the zwitterionic form and the emission

25

spectra was measured by exciting the sample at 360 nm ensures the significant protonated state population


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(Supporting information, Figure S9F). The observation of emission spectra of rGFP with similar spectral

2

features at different pH and irrespective of the excitation wavelength suggests that no excited state proton

3

transfer occurring in rGFP (Supporting information, Figure S9F-G).

4

The hydrogen bond networks stabilized the symmetric charge distribution around the chromophore residues

5

of avGFP (Arg96 & Glu222). While the incorporation of amine decreased the hydrogen bond interactions

6

in rGFP as compared to wild avGFP (Supporting Information, Figure S20A). The amine incorporation can

7

be interpreted based on it being self-zwitterionic and its stabilizing nature and thus does not require

8

additional hydrogen bonds to stabilize charge equilibration. Also, we were able to identify symmetrical Van

9

der Waals distribution around the chromophore for avGFP, and its positional deviation for the amino

10

derived variants for rGFP (Figure 3A). The Van der Waals sphere in rGFP shifts from a mean average

11

position involving a process of charge equilibration i.e., protonated amine being neutralized by a carboxylic

12

group of Glu222. Though Glu222 is known to be a substantial residue, Arg96 can also be characterized as

13

an important residue in maintaining the charge equilibration. Along with residual networks, water hydration

14

pathway also plays a vital role in relaxation dynamics. Solvent accessible sphere near chromophore is given

15

in Figure 3B. The solvent channel is highly adapted such that water channelizes through a particular path

16

to solvent accessible voids near the chromophore. It is known that Glu222 and Arg96 not only play a vital

17

role in chromophore formation but also in maintaining charge equilibrium. Based on the residual occupancy

18

distance plot it was identified that arginine had conserved contact but Glu222 changed with respect to time

19

(Figure 2B & Supporting Information, Figure S14-19).

20

3.5 rGFP maturation analysis

21

Initial spectroscopic characterization and detailed understanding of protein structure by X-ray

22

crystallographic studies and QM/MM approach emphasized investigation of red chromophore

23

transformation and maturation of rGFP, which was compared with rapidly maturing mono red fluorescent

24

protein (mRFP) variant by fluorescent measurements (Supporting Information, Figure S8). Both mRFP and


12

Page 13 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

rGFP yielded a total maturation time and a half-life (t1/2) of about ~6 and ~3 h, respectively. These results

2

demonstrated that rGFP matures faster than the Wt DsRed (t1/2~12 h) and have a maturation time very close

3

to mRuby27 (2.8 h) and mScarlet44 (2.9 h) variants of DsRed, which render the protein suitable for bio-

4

imaging applications. Hence, it can be deduced that the substitutions of NH2-Tyr in avGFP do not distort

5

the protein secondary structure (Supporting Information, Figure S3) and instead, it accelerates the protein

6

folding and maturation by intermolecular rearrangements of key residues concerning the interaction with

7

the chromophore.

8

3.6 Effect of Cryogenic temperature on rGFP by Steady state fluorescent analysis

9

The steady-state fluorescence spectra of rGFP and avGFP were measured at 100 to 400 K (Figure 4A).

10

While decreasing the temperature (310-245 K) the fluorescent intensity of avGFP was gradually diminished

11

(Figure 4A). In contrast, the fluorescence intensity of rGFP were considerably increased at their respective

12

emissions (λem = 610, 525 nm). Particularly, rGFP displayed three distinct minor peaks with the maximum

13

at 491, 521, and 570 nm upon exciting the sample at 440 nm, 100 K without suppressing the major peak at

14

610 nm. Similar phenomenon had been observed for BFP in which the dissociation of the Hydrogen bond

15

between the chromophore and His148 and strong water-mediated linkage have been attributed to the

16

enhanced fluorescent intensity at a lower temperature.45 Supporting this, rGFP also displayed strong

17

dissociation of a hydrogen bond with chromophore as compared to GFP and had strong water-mediated

18

linkage near chromophore (Figure 3B). Consequently, it enhanced the rigidity of chromophore leading to

19

increased fluorescent intensity at freezing temperature.

20

3.7 ESPT and Fluorescence Life time analysis

21

These charge equilibration and proton transfer mechanism of rGFP chromophore was demonstrated using

22

molecular simulation studies. To support this, we measured absorption and steady-state fluorescence

23

spectra of both the proteins in D2O and H2O (Figure 4B-C & Supporting Information, Figure S9). As a

24

result, we observed that the deuteration (D2O-τ1-0.97 ns, τ2-2.89 ns) of rGFP did not extend the lifetime,


13


Page 14 of 28

1

which was more or less similar to rGFP in H2O (H2O-τ1-0.955 ns, τ2-2.87 ns) (Supporting Information,

2

Table S3). To our knowledge, among the variants of DsRed, rGFP protein significantly extended the

3

average life time (3.86 ns) of red shifted protein at 610 nm as compared to DsRed variants, like mKate2

4

(2.5 ns), mRuby2 (2.5 ns), TagRFP-T (2.3 ns) and mCherry (1.5 ns).44 Similarly, mRFP consistently showed

5

biexponential decay behavior with the average fluorescence lifetime of 4.22 ns. This indicated that these

6

proteins could have acquired nearly identical but two different molecular conformations of the chromophore

7

present in the excited state, as observed in other biexponential decay fitted RFPs (eqFP611 and HcRed). To

8

explore the spectral states and electron transfer of the proteins in the presence of an electron acceptor, we

9

subjected the proteins to the fluorescent spectrum analysis in Benzoquinone (BQ) (Figure 4E-F). Increasing

10

the concentration of BQ (1.25 mM) quenched the emission of both the proteins instead of promoting the

11

electron transfer.

12

3.8 Femtosecond time-resolved transient absorption spectra

13

The femtosecond time-resolved transient absorption spectra of avGFP and rGFP in 0.1 M phosphate buffer

14

at pH 7.4 were measured by exciting the sample at 500 nm. In Figure 5A1, the transient absorption spectra

15

of avGFP consists of a positive excited-state absorption (ESA) band below 450 nm and negative band from

16

450 to 600 nm due to the overlapping ground-state bleaching (GSB) and stimulated emission (SE)

17

contributions. The transient absorption spectra decayed with the increase of spectral delay time and did not

18

attain the equilibrium even after 1.5 ns. Similarly, transient absorption spectra of rGFP were measured,

19

which have been shown in Figure 5A2. The ESA maximum was centered at around 414 nm, and a negative

20

band of GSB was observed from 450 to 600 nm. Even though, the spectral features were the same as that

21

of avGFP, in rGFP the excited state relaxation dynamics were faster and came back to equilibrium at around

22

400 ps. The kinetic decays of avGFP and rGFP at probing wavelength of 420 and 520 nm were obtained

23

by exciting at 500 nm, which are shown in Figure 5B1. The analysis of the femtosecond transient absorption

24

spectra consisting of a three-dimensional data set (wavelength, time and change in absorbance) was

25

performed with the global analysis program GLOTARAN46. Four exponential components were optimally


14

Page 15 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

fitted to the transient absorption spectra to describe the excited state dynamics completely. The transient

2

absorption spectra of avGFP were fitted with four exponentials globally and the resulting time constants

3

were τ1 = 384 fs, τ2 = 1.66 ps, τ3 = 13.26 ps, and τ4 = 3.0 ns (fixed). The time constant of 3.0 ns was fixed

4

explicitly to represent one of the components of fluorescence lifetime of avGFP obtained from TCSPC.

5

Similarly, the time constants for rGFP were τ1 = 296 fs, τ2 = 4.25 ps, τ3 = 16.06 ps, and τ4 = 234 ps. It was

6

observed that the excited state relaxation dynamics of rGFP was faster when compared to avGFP. The

7

faster dynamics in rGFP could be attributed to the changes in the conformational structure and

8

alteration in the hydrogen bonding network of the chromophore resulting from the incorporation

9

of the NH2 group compared to the avGFP and needs further investigation.

10 11

3.9 Time-resolved emission spectra

12

Time-resolved emission spectra with nanosecond time resolution were measured to investigate the excited-

13

state chemistry of avGFP and rGFP. For rGFP, the early time spectra are dominated by a rapidly decaying

14

red-shifted emission. The emission was broad, which has also been observed in the corresponding studies

15

of Discosoma sp. variant mPlum or mRFP emission (Figure 6A). For the wild-type (avGFP) there is no

16

evidence of red shift and a single band is detected with a spectrum very similar to that of the Aequorea

17

victoria emission which decays as a function of time after excitation (Supporting Information, Figure S10

18

A). Detailed analysis showed that the spectrum narrowed as a function of time after excitation in rGFP. The

19

narrowing is most apparent as subsidence of the fluorescence on the red edge at the time scale of 1.65

20

nanoseconds. However, in rGFP, no isoemissive point was observed. The TRES and time-resolved area

21

normalized emission spectra (TRANES) of rGFP, and avGFP spectrum are shown in (Figure 6B &

22

Supporting Information, Figure S10). The spectra observed for rGFP (425 and 610 nm) at 2.20 ns closely

23

matches the steady-state emission spectrum of which peaks at 610 nm. A significant observation is that the

24

TRANES intersect was not observed in GFP and rGFP variants. As reported earlier, an isoemissive point


15


Page 16 of 28

1

can only be detected in a fluorescent variant with discrete emitting states, and it disappears in a system that

2

undergoes a continuous spectral shift during emission.47,48 For rGFP, the observation of non-isoemissive

3

point indicated that the system would undergo a continuous spectral shift from native GFP to rGFP (610

4

nm) emission. Genetic incorporation of ortho NH2 induced a red shift in the fluorescence due to

5

chromophore structural modification. This is because of the interaction between the dipole of the

6

chromophore and that of the surrounding solvent molecules. The data suggest that NH2-Tyr could provoke

7

the positional shift occupancy of Glu222 and Arg96 resulting in extended conjugation and relaxed

8

chromophore, where higher solvent polarity in GFP resulted in the greater red shift.

9

4. Conclusion

10

In conclusion, this study imparts new insights into the dynamic behavior of rGFP with extended Stokes

11

shift. Earlier reports proved that the unusual red shift of RFP (TagRFP 675) attributed to the multiple

12

hydrogen bonds with the chromophore. Interestingly, in contrast to the multiple hydrogen bonding, we

13

observed a reduced number of hydrogen bonds between the chromophore and surrounding key residues.

14

Upon incorporation of the amino group, the chromophore (o-NH2p-HBI) has transformed from cis to trans

15

isomer. We observed the enhanced fluorescence lifetime and rapid, excited state relaxation in rGFP by the

16

incorporation of NH2-Tyr, which might be effective modalities for extended Stokes shift. Besides, the

17

rearrangement of the hydrogen bonding network displayed an alteration in the symmetrical Van der Waals

18

distribution around the oApHBI of rGFP as compared to the avGFP. Above all, the enhanced fluorescence

19

intensity at cryogenic temperature unraveled new avenues for the application of rGFP in newly emerging

20

technique, specifically cryogenic single molecule super-resolution imaging. These results elucidated the

21

extended Stokes shift in detail, along with designed computational and experimental methods for

22

developing FPs with new properties accounting for desired applications.

23

ASSOCIATED CONTENT

24

Supporting Information.


16

Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

General details on the material and methods, full characterization of the proteins, optimization of the

2

conditions of avGFP and rGFP biosynthesis, Crystal structure details, Biophysical characterization,

3

Bioinformatics analysis.

4

Notes

5

There are no conflicts to declare

6

ACKNOWLEDGMENT

7

Department of Science and Technology initially supported this research under Fast Track Grant

8

(SB/YS/LS-217/2013). The authors gratefully acknowledge “Science and Technology Revolution in

9

Leather with a Green Touch” (STRAIT-1.2.3) and Zero Emission Research Initiative for Solid Waste

10

(ZERIS), 12th five-year plan project under Council of Scientific and Industrial Research (CSIR). The first

11

author George A thankfully acknowledges CSIR for the award of Senior Research Fellowship. We thank

12

the Director, CSIR-CLRI for his support during the project and allowing the work to be conducted at CSIR-

13

CLRI.

14

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17 18 19 20 21 22 23 24 25 26 27 28 29 30


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1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 1. Structural representation of avGFP and rGFP proteins. (A) Surface model (1) of the

15

avGFP where sphere represents the solvent accessible spheres (SAS). (2) SAS near the

16

chromophore surface view. (B) Molecular structure of rGFP (1), avGFP (2) chromophore and

17

selected residues in their vicinity are shown; Superimposed model (3) of rGFP, an avGFP

18

chromophore and selected residues in the vicinity of the chromophores are shown. Hydrogen bonds

19

are represented as a dashed black line. (C) Electrostatic surface map of avGFP and rGFP.

20 21 22 23 24


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Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7

Figure 2. Dihedral angle and a radial plot representing residual variation. Torsional density

8

variation of ethaline linker moiety in avGFP and rGFP (A1-4) with dihedral angle (ø,τ) density

9

representing cis-trans isomerism probability in avGFP and rGFP, respectively. (B1-4) distance pair

10

principal component analysis (PCA) of the residual interatomic vector between Chromophore

11

Arg96 and Glu222, respectively.

12 13 14 15 16


23


Page 24 of 28

1 2 3

4 5 6 7 8 9 10

Figure 3. Graphical representation of avGFP and rGFP to evaluate the ESPT function. (A) Van der Waal

11

space of avGFP (1) and rGFP (2, 3) site-specific and global substitution and (B) Solvent sphere near the

12

chromophore. (C) Predicted protonation pathway of rGFP chromophore in trans-form (1) and avGFP in cis

13

form (2) significant interacting residues around the chromophore. (D) Mechanism of proposed

14

chromophore electrostatic stability.


24

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 4. (A) Temperature dependent steady-state fluorescence of GFP and rGFP at pH 7.4 from 225 to

15

325 K. (B-C) Normalized time-resolved fluorescence decay for avGFP (B) and rGFP (C) equilibrated with

16

H2O (black) and D2O (red) at 515 nm and 610 nm , respectively as obtained by fluorescence upconversion

17

spectroscopy to understand the ESPT. Excitation occurs at 1 ps on the time axis. (D) Time resolved

18

fluorescence spectroscopic analysis of avGFP, rGFP and mRFP dissolved in 50 mM Phosphate buffer pH

19

7.4. (E-F) The avGFP and rGFP were prepared at a similar concentration, equilibrated with Benzoquinone


25


Page 26 of 28

1

(BQ), excited at the 400 nm and 460 nm and monitored the fluorescence emission spectra of avGFP (E)

2

and rGFP (F) at 525 nm and 610 nm to evaluate the ESPT function.

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Figure 5. (A) Femtosecond transient absorption spectra of avGFP (1) and rGFP (2) in 0.1M phosphate

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buffer at pH 7.4 obtained by exciting at 500 nm at RT. The grey dashed line represents the steady-state


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Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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absorption spectra. (B) Kinetic profile at 420 and 520 nm obtained from femtosecond transient absorption

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spectra of avGFP (red) and rGFP (black) at pH 7.4. Inset of panel B2 shows the expanded kinetic trace at

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520 nm for both avGFP and rGFP.

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Page 28 of 28

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Figure 6. Time-resolved intensity (A) and (B) area normalized emission spectra (TRANES) of

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rGFP excited at 375 nm. Time increases in the direction of the arrow.

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TOC GRAPHICS

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Excited State Electronic Interconversion and Structural Transformation

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