title: General strategy for the bioorthogonal incorporation of strongly absorbing, solvation-sensitive infrared probes into proteins.
abstract: A high-sensitivity metal-carbonyl-based IR probe is described that can be incorporated into proteins or other biomolecules in very high yield via Click chemistry. A two-step strategy is demonstrated. First, a methionine auxotroph is used to incorporate the unnatural amino acid azidohomoalanine at high levels. Second, a tricarbonyl (η(5)-cyclopentadienyl) rhenium(I) probe modified with an alkynyl linkage is coupled via the Click reaction. We demonstrate these steps using the C-terminal domain of the ribosomal protein L9 as a model system. An overall incorporation level of 92% was obtained at residue 109, which is a surface-exposed residue. Incorporation of the probe into a surface site is shown not to perturb the stability or structure of the target protein. Metal carbonyls are known to be sensitive to solvation and protein electrostatics through vibrational lifetimes and frequency shifts. We report that the frequencies and lifetimes of this probe also depend on the isotopic composition of the solvent. Comparison of the lifetimes measured in H2O versus D2O provides a probe of solvent accessibility. The metal carbonyl probe reported here provides an easy and robust method to label very large proteins with an amino-acid-specific tag that is both environmentally sensitive and a very strong absorber.
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Ivan
authors.initials: I
authors.lastname: Peran
authors.forename: Tracey
authors.initials: T
authors.lastname: Oudenhoven
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Woys
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Watson
authors.forename: Tianqi O
authors.initials: TO
authors.lastname: Zhang
authors.forename: Isaac
authors.initials: I
authors.lastname: Carrico
authors.forename: Martin T
authors.initials: MT
authors.lastname: Zanni
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Raleigh
mesh_terms: D013058:Mass Spectrometry; D015335:Molecular Probes / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D066244:Proton Magnetic Resonance Spectroscopy; D013055:Spectrophotometry, Infrared
chemical_list: D015335:Molecular Probes; D011506:Proteins
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