Noninvasive tracing of recombinant proteins with "fluorophenylalanine-fingers".
High-level residue-specific replacement of phenylalanine residues in recombinant human annexin V and azurin from Pseudomonas aeruginosa with o-fluorophenylalanine, m-fluorophenylalanine, and p-fluorophenylalanine has been achieved using the selective pressure incorporation method. Incorporation was confirmed analytically and by UV spectroscopy while the secondary and tertiary structures of these protein mutants in solution remained unchanged upon the effected substitutions. Fluorinated phenylalanines alone and when integrated into proteins exhibit two characteristic and prominent shoulders ("fingers") in the UV spectrum in the range of 260-270 nm, which do not overlap with the contributions of tyrosine and tryptophan residues in the protein UV spectra. Thus, the presence of such "fluorophenylalanine fingers" ("FF fingers") opens a new spectral window to identify the labeled target protein among other nonlabeled cellular proteins in preparative work by simple UV spectroscopy. In the coming era of proteomics such a reliable, cheap, and easy reproducible methodology might have a great potential for speeding up the identification and characterization of target molecules in the total protein output from the genomes of a variety of organisms.
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, Martinsried, D-82152, Germany.
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Minks
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Huber
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Moroder
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Budisa
D017304:Annexin A5 / Q000378:metabolism; D001400:Azurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D005461:Fluorine / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D010649:Phenylalanine / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays; D010135:p-Fluorophenylalanine / Q000378:metabolism*
D017304:Annexin A5; D008081:Liposomes; D011994:Recombinant Proteins; D001400:Azurin; D005461:Fluorine; D010649:Phenylalanine; D010135:p-Fluorophenylalanine; D002118:Calcium