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Electron-deficient p-benzoyl-l-phenylalanine derivatives increase covalent chemical capture yields for protein-protein interactions.

ABSTRACT: The photoactivatable amino acid p-benzoyl-l-phenylalanine (pBpa) has been used for the covalent capture of protein-protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein-protein interactions. The analogs can be incorporated into live yeast and upon irradiation capture endogenous PPIs. Halogenated pBpas will extend the scope of PPIs that can be captured and expand the toolbox for mapping PPIs in their native environment.

SUBMITTER: Joiner CM

PROVIDER: S-EPMC6511736 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Electron-deficient p-benzoyl-l-phenylalanine derivatives increase covalent chemical capture yields for protein-protein interactions.

Joiner Cassandra M CM Breen Meghan E ME Mapp Anna K AK

Protein science : a publication of the Protein Society 20190429 6

The photoactivatable amino acid p-benzoyl-l-phenylalanine (pBpa) has been used for the covalent capture of protein-protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein-protein interactions. The analogs can be incorporated into live yeast ...[more]

PMID: 30977234

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Project description:In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds.

Dataset Information

Electron-deficient p-benzoyl-l-phenylalanine derivatives increase covalent chemical capture yields for protein-protein interactions.

Publications

Electron-deficient p-benzoyl-l-phenylalanine derivatives increase covalent chemical capture yields for protein-protein interactions.

Similar Datasets

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