Site-specific independent double labeling of proteins with reporter atoms

Many types of physical, spectroscopic, and biological studies of proteins a nd other macromolecules are facilitated by the incorporation of reporter gr oups. In many cases these are single atom substitutes, for example isotopes (C-13 for C), or light (F for H) and heavy (Se for S) atom homologs. In so me circumstances the incorporation of two different labels in the same mole cule would be greatly desirable. Commonly used protein engineering methods for incorporating them can rarely cope with differential double labeling, a nd have other limitations such as universal, non-specific, or random incorp oration. Although de novo peptide synthesis has the power to achieve highly specific labeling, the difficulties inherent in creating long sequences le ad us to propose protein semisynthesis as the most practical approach. By l igating combinations of natural and labeled synthetic fragments to reform h oloproteins, we can overcome any of the limitations discussed. Using cytoch rome c as a model protein we show that two reporter atoms, selenium and bro mine, can be simultaneously and site-specifically incorporated without sign ificant consequences to structure and (or) function. This capability opens up the prospect of advances in a number of areas in structural biology.