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Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA

(RECEIVED July 2, 2004; FINAL REVISION August 30, 2004; ACCEPTED August 30, 2004)

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys₂His₂ zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.

Keywords: covalent modification; biosensor; fluorescence resonance energy transfer; surface immobilization

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04965405.

Reprint requests to: Homme W. Hellinga, Duke University Medical Center, Department of Biochemistry, Box 3711, Research Drive, 415 Nanaline Duke Building, Durham, NC 27710, USA; e-mail: hwh{at}biochem.duke.edu; fax: (919) 684-8885.

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