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Area of Science:

  • Biochemistry
  • Structural Biology
  • Spectroscopy

Background:

  • Understanding local protein environments is crucial for protein function and engineering.
  • Unnatural amino acids (UAAs) offer unique reporter capabilities for probing protein structure.
  • 4-cyano-l-phenylalanine (pCNPhe) serves as a vibrational reporter for studying protein microenvironments.

Purpose of the Study:

  • To develop and apply a multifaceted approach for investigating complex local protein environments.
  • To utilize the vibrational reporter UAA, pCNPhe, within the superfolder green fluorescent protein (sfGFP) model system.
  • To provide a molecular interpretation of the local environment surrounding pCNPhe at specific sites within sfGFP.

Main Methods:

  • Developed a two-step enantioselective synthesis for high-yield, chromatography-free production of pCNPhe.
  • Genetically incorporated pCNPhe at three distinct sites (74, 133, 149) in sfGFP using Amber codon suppression and an orthogonal tRNA synthetase in *E. coli*.
  • Combined temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to analyze the protein-UAA interactions.

Main Results:

  • Successfully synthesized pCNPhe with 87% yield and high purity.
  • Site-specifically incorporated pCNPhe into sfGFP at surface (133, 149) and interior (74) locations.
  • Characterized site 133 as solvent-exposed, site 149 as partially buried, and site 74 as having three distinct local environments (van der Waals, water H-bonding, histidine H-bonding).

Conclusions:

  • The integrated approach of IR spectroscopy, X-ray crystallography, and MD simulations effectively probes local protein environments using UAAs.
  • pCNPhe incorporation and analysis provide detailed molecular insights into the microenvironments of specific amino acid residues within proteins.
  • This methodology is valuable for understanding protein structure-function relationships and for protein engineering efforts.