Unnatural Amino Acid Mutagenesis of Green Fluorescent Protein

2003 ◽  
Vol 68 (1) ◽  
pp. 174-176 ◽  
Author(s):  
Lei Wang ◽  
Jianming Xie ◽  
Ashok A. Deniz ◽  
Peter G. Schultz
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Unnatural Amino Acid ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
Green Fluorescent

scholarly journals Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine

2018 ◽  
Vol 74 (10) ◽  
pp. 650-655
Author(s):  
Nicole Maurici ◽  
Nicole Savidge ◽  
Byung Uk Lee ◽  
Scott H. Brewer ◽  
Christine M. Phillips-Piro
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Structure ◽  
Amino Acid ◽  
Crystal Structures ◽  
Fluorescent Protein ◽  
Unnatural Amino Acid ◽  
Asymmetric Unit ◽  
Space Group ◽  
Green Fluorescent ◽  
Amber Codon Suppression

The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-L-phenylalanine (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3221 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNO2F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4122 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNO2F or Asn149pNO2F sfGFP with wild-type sfGFP resulted in small root-mean-square deviations, illustrating that these residues do not significantly alter the protein structure and supporting the use of pNO2F as an effective spectroscopic reporter of local protein structure and dynamics.

scholarly journals Assessing Local Environments in Green Fluorescent Protein using Unnatural Amino Acid 4‐Cyano‐L‐Phenylalanine

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Brianna Papoutsis ◽  
ByungUk Lee ◽  
Scott Brewer ◽  
Christine Phillips‐Piro
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Unnatural Amino Acid ◽  
Local Environments ◽  
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.

scholarly journals Unraveling Complex Protein Environments in Green Fluorescent Protein using the Unnatural Amino Acid 4-cyano-L-phenylalanine

2020 ◽  
Vol 118 (3) ◽  
pp. 39a
Author(s):  
Brianna M. Papoutsis ◽  
ByungUk Lee ◽  
Nathan Wong ◽  
Paul Nerenberg ◽  
Scott H. Brewer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Unnatural Amino Acid ◽  
Complex Protein ◽  
Green Fluorescent

scholarly journals Probing Structural Implications of Unnatural Amino Acid Incorporation into Green Fluorescent Protein

2015 ◽  
Vol 108 (2) ◽  
pp. 511a
Author(s):  
Nicole Maurici ◽  
Andrew Dippel ◽  
Melanie Liskov ◽  
Scott Brewer ◽  
Christine Phillips-Piro
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Green Fluorescent

In Vivo Photocrosslinking with Unnatural Amino Acid Mutagenesis

ChemBioChem ◽  
2002 ◽  
Vol 3 (11) ◽  
pp. 1135-1137 ◽  
Author(s):  
Jason W. Chin ◽  
Peter G. Schultz
Keyword(s):  
Amino Acid ◽  
Unnatural Amino Acid ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis

Unnatural amino acid mutagenesis in mapping ion channel function

2003 ◽  
Vol 13 (3) ◽  
pp. 264-270 ◽  
Author(s):  
Darren L Beene ◽  
Dennis A Dougherty ◽  
Henry A Lester
Keyword(s):  
Amino Acid ◽  
Ion Channel ◽  
Unnatural Amino Acid ◽  
Channel Function ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis

scholarly journals Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations

2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Shu-su Liu ◽  
Xuan Wei ◽  
Xue Dong ◽  
Liang Xu ◽  
Jia Liu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Structural Plasticity ◽  
Green Fluorescent
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