Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: Overview and feasibility: Properties of diazirine as a precursor

Authors

  • Frederic M. Richards,

    Corresponding author
    1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
    • Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, New Haven, Connecticut 06520-8114
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  • Raphael Lamed,

    1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
    Current affiliation:
    1. Department of Molecular Biology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel
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  • Richard Wynn,

    1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
    Current affiliation:
    1. Department of Applied Biotechnology, The DuPont Pharmaceutical Co., Experimental Station, P.O. Box 80336, E336/241B, Wilmington, Delaware 19880-0336
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  • Darshan Patel,

    1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
    Current affiliation:
    1. 1025 Walnut St., Box 576, Philadelphia, Pennsylvania 19107-5001
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  • Gerard Olack

    1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
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Abstract

Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra : CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.

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