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

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

Journal of Biological Engineering ◽

10.1186/s13036-016-0031-6 ◽

2016 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Georg Wandrey ◽

Joel Wurzel ◽

Kyra Hoffmann ◽

Tobias Ladner ◽

Jochen Büchs ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Amino Acid ◽

High Throughput ◽

High Throughput Screening ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Unnatural Amino Acid ◽

Genetic Code Expansion ◽

Screening Platform ◽

Green Fluorescent

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Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein

10.1101/079061 ◽

2016 ◽

Author(s):

Eleisha L. Jackson ◽

Stephanie J. Spielman ◽

Claus O. Wilke

Keyword(s):

Green Fluorescent Protein ◽

Protein Structure ◽

Amino Acid ◽

Protein Design ◽

Fluorescent Protein ◽

Computational Prediction ◽

Single Amino Acid ◽

Dimensional Structure ◽

Amino Acid Deletion ◽

Green Fluorescent

AbstractProteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single–amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions.

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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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