Protein footprinting

The interacting ligand producing the footprint may be DNA,13,20 RNA (Fig. 4.1416), or protein.17,18,21,22 The procedure below focuses on protein-RNA interactions but it is applicable for several other types of ligands. 

Bacterial expression vectors developed for protein footprinting 

A common feature of all expression plasmids is the opposite positions of the purification- and kinase tags. See Section 3.2 for expression procedures. 

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. 

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Greiner, D.P., Hughes, K.A., Gunasekera, A.H., and Meares, C.F. (1996) Binding of the sigma-70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting. Proc. Natl. Acad. Sci. USA 93, 71-75. 

Xu, G. Chance, M.R. Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting. Anal. Chem. 2005, 77, 4549-4555. 

Heyduk, E. and Heyduk, T. (1994) Mapping protein domains involved in macromolecular interactions a novel protein footprinting approach. Biochemistry 33, 9643-9650. 

Takamoto K, Chance MR. (2006) Radiolytic protein footprinting with mass spectrometry to probe the structure of macromolecular complexes. Annual Review of Biophysics and Biomolecular Structure 35 251-276. 

Interaction of the functionalized-monolaycr electrode with Fe(III)-protoporphyrin IX, (15), resulted in the reconstitution of the protein with the two heme units. The surface coverage of the reconstituted de novo hemoprotein was found to be 2.5x10 mole cm Assuming that the protein footprint area is ca. 25 A, the saturated surface coverage of the de novo protein corresponds to 3.5x 10" mole cm and thus the experimental value... 

When producing proteins aimed for protein footprinting experiments (see Section 4.5) the purity, rather than the quantity of the expressed protein, is critical. By positioning the affinity-tag and the phosphorylation site at opposite ends of the introduced cDNA, only full-length protein becomes radiolabelled after purification14 (see Table 4.4 in Chapter 4). 

Jensen, T.H., Leffers, H. and Kjems, J. (1995). Intermolecular binding sites of HIV-1 Rev protein determined by protein footprinting. J. Biol. Chem. 270, 13777-13784. 

Fig. 4.13. Flow chart of the protein footprinting technique. Purified recombinant protein, which has been radioactively labelled at the N- or C-terminal, is cleaved by proteinases or chemicals with single-hit kinetics. The substrate is kept at native conditions in uncomplexed and complexed form. The cleavage products are analysed in high resolution SDS gels.

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...

Label a tube for each protein footprinting reaction (or use a microtiter plate). 

To enhance the resolution of small protein products we recommend Tris/Tricine-SDS-polyacrylamide gels24 for the protein footprinting... 

Jensen, T.H., Jensen, J., Szilvay, A.M. and Kjems, J. (1997). Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody binding sites. FEBS Lett. 414, 50-54. 

Lykke-Andersen, J.. Garrett, R.A. and Kjems. J. (1996). Protein footprinting approach to mapping DNA binding sites of two archael homing enzymes evidence for a two-domain protein structure. Nucleic Acids Res. 24, 3982-3989. 

Heyduk, T., Heyduk, E., Severinov, K., Tang, H. and Ebrigth, R.H. (1996). Determinants of RNA polymerase a subunit for interaction with /3, /3 and cr subunits Hydroxyl-radical protein footprinting. Proc. Natl. Acad. Sci. USA 93, 10162-10166. 

Fig. 10. Protein and DNA footprints (Jones et al., 1999). The protein footprint differentiates between the residues contacting the sugar-phosphate backbone and those contacting the bases. Protein residues that make no contact with the DNA are colored blue. Those connecting the sugar-phosphate backbone are colored red, and those making base contacts are colored yellow, (a) Proteins with a single binding head (b) proteins with a double binding head and (c) proteins with an envelope mode of binding.

MS-based strategies based on cross-linking and protein footprinting provide alternative approaches for epitope mapping [31]. Although these methods cannot reveal three-dimensional structures of antibody-antigen complexes, the information on the location of both linear and conformational epitopes can be obtained. In addition, the sample amount required for MS measurements is small (femtomole to picomole), and there is no known upper mass limit when a proteolytic step is included. 

The protein footprinting approach utilizes chemical modifications to generate a mass shift either on amino acid side chains or the protein amide backbone, followed by MS analysis. This approach can capture minor changes in protein solvent accessibilities that are induced by protein-ligand (ligand small molecule, protein, DNA, etc.) interactions or protein conformational changes. 

Gau, B.C., Sharp, J.S., Rempel, D.L., Gross, M.L. (2009) East Photochemical Oxidation of Protein Footprints Faster than Protein Unfolding. Anal. Chem. 81 6563-6571. 

Considering the size of a protein (footprint w 20 nm ) and the fact that proteins adsorb via multiple sites, we estimate that at least 10 PEG chains directly contribute to the adsorption barrier of a single protein. Therefore, it takes 2 kT per... 

Protein Footprinting, Cross-linking, and Surface Labeling. 135... 

Protein Footprinting, Cross-Linking, and Surface Labeiing... 

The principle of protein footprinting rehes on differences in the enzymatic proteolysis rate of proteins that closely interact with other molecules or not. The conceptual idea is that certain parts of their primary structure are protected (shielded) from proteolysis by close interactions with other molecules, with the consequence that the protease used will not or less efficiently catalyze cleavages within these regions (Fig. 10). As any cleavage, independent of where, can destabilize the interaction complex because it shortens or disrupts the primary structure of the protein, a major risk for protein footprinting is that the structure in question is quickly altered during the course of the reaction. Whether this happens early on, at a late state or not, is usually unknown. Therefore, the reaction is monitored over time, or the concentration of the protease is varied in parallel experiments [ 164-168]. 

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