scholarly journals Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids

Molecules ◽  
2017 ◽  
Vol 22 (7) ◽  
pp. 1194 ◽  
Author(s):  
Jordan K. Villa ◽  
Hong-Anh Tran ◽  
Megha Vipani ◽  
Stephanie Gianturco ◽  
Konark Bhasin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Fluorescence Modulation ◽  
Green Fluorescent

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Gregory M. Olenginski ◽  
Juliana Piacentini ◽  
Darcy R. Harris ◽  
Nicolette A. Runko ◽  
Brianna M. Papoutsis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Unnatural Amino Acid ◽  
Green Fluorescent ◽  
Absorbance Band ◽  
Protein Constructs

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

Reversible biomechano-responsive surface based on green fluorescent protein genetically modified with unnatural amino acids

2015 ◽  
Vol 51 (1) ◽  
pp. 232-235 ◽  
Author(s):  
Johan Longo ◽  
Chunyan Yao ◽  
César Rios ◽  
Nguyet Trang Thanh Chau ◽  
Fouzia Boulmedais ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Genetically Modified ◽  
Uniaxial Stretching ◽  
Green Fluorescent

Uniaxial stretching of modified GFP “clicked” onto an elastomer leads to a repeatable and reversible decrease of its fluorescence intensity.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

2014 ◽  
Vol 44 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Akio Kawahara-Kobayashi ◽  
Mitsuhiro Hitotsuyanagi ◽  
Kazuaki Amikura ◽  
Daisuke Kiga
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Experimental Evolution ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study

2016 ◽  
Vol 72 (1) ◽  
pp. 121-130 ◽  
Author(s):  
Andrew B. Dippel ◽  
Gregory M. Olenginski ◽  
Nicole Maurici ◽  
Melanie T. Liskov ◽  
Scott H. Brewer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Structure ◽  
Crystal Structures ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Local Environment ◽  
Loop Region ◽  
Selective Placement ◽  
Green Fluorescent ◽  
Local Protein

The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-L-phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent-exposed loop region and/or a partially buried site on the β-barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made. The structural implications were quantified by comparing the root-mean-square deviation (r.m.s.d.) between the crystal structure of wild-type sfGFP and the protein constructs containing either pCNF or pCCF in the local environment around the UAAs and in the overall protein structure. The results suggest that the selective placement of these spectroscopic reporter UAAs permits local protein environments to be studied in a relatively nonperturbative fashion with site-specificity.

scholarly journals The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies

2002 ◽  
Vol 366 (2) ◽  
pp. 491-500 ◽  
Author(s):  
Frank DIETZ ◽  
Sebastian FRANKEN ◽  
Kenya YOSHIDA ◽  
Hideji NAKAMURA ◽  
Joachim KAPPLER ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Factor Activity ◽  
Hek 293 ◽  
Green Fluorescent

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1μg/ml. The expression of the fusion proteins, bHRP-4–green fluorescent protein and mHDGF–green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42nM. Comparison of the bHRP-4 amino acid sequence with HRP-1–3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

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