Protein footprinting using proteinases

A large collection of endoproteinases is available and they are crudely divided into two main groups, the specific and unspecific proteinases, depending on their level of specificity (summarised in Table 4.5). The specific proteinases, in general, yield few cleavages 

Endoproteinase Type Primary specificity Comments Suggested concentration 

Proteinase K Serine P-1 1 Pi P-1 = non-specific but aromatic or hydrophobic amino acids preferred. Pi = non-specific. 5-50 pg/p.1 

Subtilisin Carlsbergensis Serine p 11 Pi P—i = non-specific but neutral and acidic amino acids, p, = non-specific 0.5-5 ng/p.1 

32P-labelled protein (5 ng/jxl in complex buffer)—see Section 3.2.4 for preparation. 

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Both endoproteinases and chemicals can be used to cleave the protein, provided they are active at conditions optimal for complex formation. Table 4.5 provides a list of 13 commercially available proteinases which are useful for protein footprinting. The majority of chemicals reactive towards proteins suffers from the drawback of being reactive towards nucleic acids as well. Hydroxyl radicals produced from H202 in the vicinity of Fe2+ ions are, however, useful for protein surface predictions and for mapping DNA-13 and RNA binding sites (Fig. 4.1423) on proteins. 

Fig. 4.14. Autoradiogram of a protein footprinting gel. Protein footprinting of poly(rC) (RNA) on the N-terminal KH domain of PCBP1 (protein) using hydroxyl radical cleavage (Chem), proteinases as indicated and no reagent (Con). The protein binds specifically to poly(rC) but not to poly(rG). Cleavages that are either inhibited or enhanced by poly(rC) are indicated by filled and open arrows, respectively. See Leffers et al. for details.23...

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