Protease mapping

Transbilayer movement of lipid at the endoplasmic reticulum In eukaryotic systems a detailed pattern of synthetic asymmetry has emerged with respect to the topology of the enzymes of phospholipid synthesis in rat liver microsomal membranes. Protease mapping experiments (D.E. Vance, 1977 R. Bell, 1981) have indicated that the active sites of the phospholipid synthetic enzymes are located on the cytosolic face of the ER. Thus, in both prokaryotic and eukaryotic systems, it appears that the site of synthesis of the bulk of cellular phospholipid is the cytosolic side of the membrane. This asymmetric localization of synthetic enzymes strongly implicates transbilayer movement of phospholipids as a necessary and important event in membrane assembly that is required for the equal expansion of both leaflets of the bilayer [13]. 

Protease mapping is a convenient method of identifying proteins and pol eptides, requiring no special equipment or technical facility beyond that necessary for SDS-polyacrylamide slab gel electrophoresis. The technique is performed mainly in two different ways. 

The common proteases used in protease mapping are chymotrypsin. Staphylococcus aureus protease, papain, subtilisin, Streptomyces griseus protease, flcln, and elastase. Of these, papain requires 2-mercaptoethanol which inhibits acrylamide polymerization. This protease therefore cannot be used for proteolysis during electrophoresis. Cofactors needed by proteases can be incorporated into the gels. 

Keidel S, LeMotte P, Apfel C (1994) Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping. Mol Cell Biol 14 287-298... 

Lamphear, B. J., Kirchweger, R., Skem, T., and Rhoads, R. E. (1995). Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J. Biol. Chem. 270, 21975—21983. 

Ghaim, J.B., Greiner, D.P., Meares, C.F., and Gennis, R.B. (1995) Proximity mapping the surface of a membrane protein using an artificial protease demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd. Biochemistry 34(36), 11311-11315. 

Owens, J.T., Miyake, R., Murakami, K., Chmura, A.J., Fujita, N., Ishihama, A., and Meares, C.F. (1998) Mapping the sigma(70) subunit contact sites on Escherichia coli RNA polymerase with a sigma(70)-conjugated chemical protease. Proc. Nat. Acad. Sci. USA 95, 6021-6026. 

Fig. 6. Terminal capping and lateral bulging of globular domains in the //-solenoid of the hemoglobin protease from E. coli (Otto et al., 2005). The //-solenoid domains are shown in blue and the remaining regions in dark yellow. (A) Ribbon diagram of the 3D structure and (B) linear map of the domain distribution within the amino acid sequence.

The primary analytical applications of RPLC in the development of biopharmaceuticals are the determination of protein purity and protein identity. Purity is established by analysis of the intact protein, and RPLC is useful in detecting the presence of protein variants, degradation products, and contaminants. Protein identity is most often established by cleavage of the protein with a site-specific protease followed by resolution of the cleavage products by RPLC. This technique, termed peptide mapping, should yield a unique pattern of product peptides for a protein that is homogeneous with respect to primary sequence. 

A variety of proteases are used for peptide mapping, but trypsin is the most popular because it is stable and available in high purity with little contamination... 

Interpretation of the electron density maps showed that the large subunit could not be modelled beyond His536 (Fig. 6.10), that is fifteen amino acids short of the 551 residues predicted by the nucleotide sequence (Table 6.2). At about the same time, the cleavage of this fifteen-residue stretch, which is performed by a specific protease, was reported to be an obligatory step for the maturation of the enzyme (Menon et al. 1993). It is also of interest to note that in all [NiFe] hydrogenase crystal structures this buried C-terminal histidine is ligated to a metal atom which is either a magnesium or an iron (see above). 

The hydrophobic peptide segments of El and E2, which attach the spike protein to the lipid bilayer, can be localized on the polypeptide chains by a mapping procedure first used by Dintzis (1961) to show that the synthesis of polypeptide chains begins at the amino-terminal end. The hydrophobic stubs left in the viral membrane after protease treatment are found at the carboxyl-terminal ends of both the El and the E2 polypeptides (Garoff and Sdderlund, 1978). 

Schlosser, A., Vanselow, J.T. and Kramer, A. (2005) Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Analytical Chemistry, 77, 5243-5250. 

In the first study, Charifson and coworkers (16) performed virtual ligand screening on p38 MAP kinase, IMPDH, and HIV protease. For each of these targets, they chose 400 or more test compounds in three activity ranges, and... 

The cysteinyl proteases include papain calpains I and II cathepsins , H, and L proline endopeptidase and interleukin-converting enzyme (ICE) and its homologs. The most well-studied cysteinyl protease is likely papain, and the first x-ray crystallographic structures of papain [193] and a peptide chloromethylketone inhibitor-papain complex [194] provided the first high resolution molecular maps of the active site. Pioneering studies in the discovery of papain substrate peptide-based inhibitors having P, electrophilic moieties such as aldehydes [195], ketones (e.g., fluoromethylketone, which has been determined [196] to exhibit selectivity for cysteinyl proteases versus serinyl proteases), semicarbazones, and nitriles are noteworthy since 13C-NMR spectro-... 

A variation on the theme has been to map out protease specificity.28 A library of fusion proteins was constructed in a modular manner. The synthetic protein had an N-terminal domain that binds very tightly to an affinity column. This domain was connected to the C-terminal domain of M13 gene III by a randomized peptide sequence. The phages were then bound to the affinity support and treated with a protease. Phages that had a protease-susceptible site were cleaved from the support and eluted. This procedure was subsequently used to map out the specificity of furin,29 which is described in the next chapter. 

HIV-1 genetic resistance to protease inhibitors occurs via specific mutations. Genotypic analysis of the HIV protease gene from isolates selected in vitro indicated that Gly48Val and Leu90Met mutants had reduced susceptibility to saquinavir (Ohta, 1997). Indinavir and ritonavir resistance maps to residue 82, whereas for amprenavir the key mutation is at residue 50 (I50V) and confers a threefold decline in viral sensitivity to amprenavir. Two additional mutations at residues 46 and 47 follow development of mutation at position 50, resulting in a 20-fold de-... 

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