Footprinting reagents

Several metal complexes now serve as high-resolution, sequence-neutral chemical footprinting reagents. Some of these reagents are shown in Figure 8.13. The first, as mentioned previously, was MPE-Fe(II). The complex con-... 

In order to function as an effective footprinting reagent, a metal complex must be a relatively nonspecific DNA cleaving agent. The presence of a... 

The lack of any direct contact between the metal complex and DNA is a unique advantage for [Fe(EDTA)] footprinting. Any molecule that binds to DNA will have some effect on its structure. For example, footprinting reagents that bind by intercalation unwind the DNA helix and this may change the observed binding site or orientation of a protein. Since [Fe(EDTA)]- does not bind, we can be fairly sure that the footprint observed is not influenced by structural distortions introduced by the probe molecule. 

The sequence neutrality for random stretches of double-stranded DNA makes uranyl ion a very useful reagent for examining contact regions in protein-DNA complexes. Such photo-footprinting studies have been carried out with the A-repressor/ORl [185], E. Coli RNA polymerase/deo Plpromoter [187] and transcription factor IIIA-ICR [188]. 

Chemical reagents have been used in solution to footprint sites of interaction in noncovalent complexes. Only a limited set of studies have been performed in the gas phase using ion—molecule reactions between a charged noncovalent complex and various neutral reagents [72,73]. They can be classified into two areas H/D exchange reactions [72] and ligand switching reactions [73]. Recent results in these two areas are discussed below. 

Several crystallization-screening protocols have been developed over the past decade to help identify initial crystallization conditions. These include footprint screening (Sutra et al., 1992), sparse matrix sampling (Jancarik and Kim, 1991), statistical methods (Carter and Carter, 1979), and MON-48 (Shieh et al., 1991). Reagent kits for some of these screens are now commercially available (Hampton Research [www.hamptonresearch.com], Emerald BioStructures [www.emeraldbio-structures.com]) and are commonly used in the laboratory. 

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