Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

Green fluorescent protein fusions were constructed with several oxidative stress promoters from Escherichia coli. These promoters were chosen for their induction by reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals. When exposed to various free radical insults, the cells fluoresced with great specificity based on the corresponding ROS. In this work, we propose a way in which these constructs could be used to study the mode of action of a variety of antitumor drugs. This approach offers the possibility of complementing gene chip technology by the creation of living chips for high throughput screening as well as studying differential gene expression.

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Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.12.3682-3687.2003 ◽

2003 ◽

Vol 47 (12) ◽

pp. 3682-3687 ◽

Cited By ~ 177

Author(s):

Chartchai Changsen ◽

Scott G. Franzblau ◽

Prasit Palittapongarnpim

Keyword(s):

Green Fluorescent Protein ◽

High Throughput ◽

High Throughput Screening ◽

Antimicrobial Agents ◽

Fluorescent Protein ◽

Signal To Noise Ratio ◽

Enhanced Fluorescence ◽

Signal To Noise ◽

Green Fluorescent ◽

Fluorescent Protein Reporter

ABSTRACT The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

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

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1801169x ◽

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.

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

The FASEB Journal ◽

10.1096/fasebj.2021.35.s1.04055 ◽

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

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

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321006525 ◽

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.

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Application of Green Fluorescent Protein-Based Chloride Indicators for Drug Discovery by High-Throughput Screening

Reviews in Fluorescence 2004 ◽

10.1007/978-0-306-48672-2_6 ◽

2004 ◽

pp. 85-98

Author(s):

A. S. Verkman ◽

Peter M. Haggie ◽

Luis J. V. Galietta

Keyword(s):

Green Fluorescent Protein ◽

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Green Fluorescent

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Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471401267x ◽

2014 ◽

Vol 70 (8) ◽

pp. 2152-2162 ◽

Cited By ~ 6

Author(s):

James A. J. Arpino ◽

Pierre J. Rizkallah ◽

D. Dafydd Jones

Keyword(s):

Green Fluorescent Protein ◽

Amino Acid ◽

Long Range ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Core Protein ◽

Single Amino Acid ◽

Amino Acid Deletion ◽

Deletion Mutations ◽

Green Fluorescent

Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFPD190Δcontaining a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFPA227Δrevealed that a `flipping' mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes inBfactors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

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