title: Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.
abstract: 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.
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Shengxi
authors.initials: S
authors.lastname: Chen
authors.forename: Nour Eddine
authors.initials: NE
authors.lastname: Fahmi
authors.forename: Chandrabali
authors.initials: C
authors.lastname: Bhattacharya
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Yuguang
authors.initials: Y
authors.lastname: Jin
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D055162:Biocatalysis; D001713:Biphenyl Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D055598:Chemical Phenomena; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D005493:Folic Acid Antagonists / Q000494:pharmacology; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D007536:Isomerism; D008727:Methotrexate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014295:Trimethoprim / Q000494:pharmacology
chemical_list: D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005493:Folic Acid Antagonists; D007202:Indicators and Reagents; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010649:Phenylalanine; D014295:Trimethoprim; C479919:FolM protein, E coli; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate