Protein sequences are usually determined by sequencing the corresponding cDNA. Although this approach is efficient, it is unable to account for posttransla-tional covalent modifications such as the oxidation of cysteines forming disulfide bridges. In the era of genome-wide sequencing projects the insufficiency of this type of information is becoming more apparent. Statistics on recently sequenced organisms indicate that the function of 22-60% of putative reading frames is unknown.1,2 To reveal the full covalent structure of a protein, either the expressed or extracted protein must be analyzed experimentally or its structure needs to

1 M. A. Marti-Renom, A. Stuart, A. Fiser, R. Sánchez, F. Melo, and A. Sali, Annu. Rev. Biophys.

Biomol. Struct. 29,291 (2000).

2 J. Cedano, P. Aloy, J. A. Pérez-Pons, and E. Querol, J. Mol. Biol. 266,594 (1997).

a cysteine at the critical position 28 (see Fig. 1). In agreement with the DNA-binding experiments, coexpression of VP16/Ref-l with an N-terminal HIF-la fusion (GalDBD/HIF- la 1-245) resulted in no increase in reporter gene activity.

We believe the major advantages of analyzing redox protein interactions with the mammalian two-hybrid system are that (1) it is a cell-based in vivo interaction assay, which is more physiologically relevant than an in vitro interaction assay; and (2) it is often more sensitive than other assays such as coimmunoprecipitation, this being important because most redox interactions tend to be weak and transient in nature. Although other methods to observe transient interactions between redox proteins exist, for example, solution nuclear magnetic resonance analysis of the interaction between thioredoxin and Ref-1,13 the mammalian two-hybrid approach is by comparison simple, quick, and inexpensive, and requires no specialized expertise.

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