A General Strategy for the Bio-orthogonal Incorporation of Strongly Absorbing, Solvation Sensitive Infrared Probes into Proteins.

TitleA General Strategy for the Bio-orthogonal Incorporation of Strongly Absorbing, Solvation Sensitive Infrared Probes into Proteins.
Publication TypeJournal Article
Year of Publication2014
AuthorsPeran I, Oudenhoven TA, Woys AM, Watson MD, Zhang TO, Carrico I, Zanni MT, Raleigh DP
Secondary TitleThe Journal of Physical Chemistry B
Volume118
Issue28
Pagination7946–7953
Date Published04/2014
Abstract

A high sensitivity metal carbonyl based IR probe is described which can be incorporated into proteins or other bio-molecules 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 (η5-cyclopentadienyl) rhenium(I) probe modified with a 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 K109, 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. In this paper, 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.

URLhttp://pubs.acs.org/doi/abs/10.1021/jp5008279
DOI10.1021/jp5008279
Short TitleJ. Phys. Chem. B