Excited State Electronic Interconversion and Structural Transformation of Engineered Red-Emitting Green Fluorescent Protein Mutant

Abstract

Red fluorescent proteins with a large Stokes shift offer a limited autofluorescence background and are used in deep tissue imaging. Here, by introducing the free amino group in Aequorea victoria, the electrostatic charges of the p-hydroxybenzylidene imidazolinone chromophore of green fluorescent protein (GFP) have been altered resulting in an unusual, 85 nm red-shifted fluorescence. The structural and biophysical analysis suggested that the red shift is due to positional shift occupancy of Glu222 and Arg96, resulting in extended conjugation and a relaxed chromophore. Femtosecond transient absorption spectra exhibited that the excited state relaxation dynamics of red-shifted GFP (rGFP) (τ4 = 234 ps) are faster compared to the A. victoria green fluorescent protein (τ4 = 3.0 ns). The nanosecond time-resolved emission spectra of rGFP reveal the continuous spectral shift during emission by a solvent reorientation in the chromophore. Finally, the molecular dynamics simulations revealed the rearrangement of the hydrogen bond interactions in the chromophore vicinity, reshaping the symmetric distribution of van der Waals space to fine tune the GFP structure resulting from highly red-shifted rGFP.

Devising Efficient Red‐Shifting Strategies for Bioimaging: A Generalizable Donor‐Acceptor Fluorophore Prototype

“…By introducing noncanonical amino acids into the autocatalytically matured chromophore, the FPs’ properties such as color and FQY can be tuned. For the GFP family and its derivatives, the readily changeable chromophore moiety is the P‐ring from the tyrosine residue, and some recent works demonstrated the potential in red‐shifting GFP emission without significantly enlarging the conjugated chromophore . In terms of color, the redder GFP chromophore structure is in line with the “double donor” (an EDG next to −OH/‐O − ) structure in this review (Figure ).…”

Enhanced Cellular Uptake and Sustained Transdermal Delivery of Collagen for Skin Regeneration

“…This knowledge helps to add functional groups in the protein through unnatural amino acids to create accurately designed structural and functional proteins. These manually customized protein products have an altered structure, function, , pKa, and redox potential and could be used for protein cross-linking, cell labeling, sensor applications, and protein conjugation . In recent years, the introduction of unnatural amino acid into the enzymes for its potential applications has been increasingly investigated. , In this study, we used active hydrolyzing enzyme (α-1,4-glycosidic hydrolase–amylase), which could act on the skin collagen bundles connected by internal α - 1,4-glycosidic linkages between the adjacent monosaccharides.…”

Self‐Assembly and Mechanical Properties of Engineered Protein Based Multifunctional Nanofiber for Accelerated Wound Healing

“…Here, we generated a mutant fluorescent protein (GFPDOPA) through mis-aminoacylation or selective pressure approach, [43,44] for which Escherichia coli tyrosine auxotrophic cells were transformed with a plasmid pQE80-GFP and were grown overnight in minimal media containing 20 amino acids (40 mg mL −1 ). The same cells were transferred to minimal media with 19 amino acids (40 mg L −1 ) and a limited concentration of L-tyrosine (0.03 mm).…”

“…As a result, eight tyrosine (Y) residues in the GFP including chromophore residue (Y66) were replaced with DOPA and was purified by affinity chromatography from the same expression host. [43,44] The quantitative substitution of tyrosine by DOPA in GFP was detected by MALDI-TOF analysis with a determined mass of 28 394 Da when compared to the mass of GFP (28°64 Da). (Figure S1C,D, Supporting Information).…”

“…From this result, we assumed that DOPA‐boric acid complexation had taken place on the surface exposed DOPA residues at Y92, Y151, Y182, Y200, and Y273 in GFP as described in an earlier report. [ 44 ] The pH‐dependent reversible conjugation between GFPDOPA and BA was further characterized by using 11 B NMR spectroscopy and was compared with a pure BA (Figure 4G). A sharp peak at −19 ppm corresponded to boric acid conjugated with GFPDOPA (Figure 4G), which confirmed that boric acid forms a GFPDOPA‐boronate complex by interacting with the OH group of GFPDOPA.…”