Cleavage sites

Asp 189 at the bottom of the substrate specificity pocket interacts with Lys and Arg side chains of the substrate, and this is the basis for the preferred cleavage sites of trypsin (see Figures 11.11 and 11.12). It is almost trivial to infer, from these observations, that a replacement of Asp 189 with Lys would produce a mutant that would prefer to cleave substrates adjacent to negatively charged residues, especially Asp. On a computer display, similar Asp-Lys interactions between enzyme and substrate can be modeled within the substrate specificity pocket but reversed compared with the wild-type enzyme. 

This polylinker contains restriction sites for BamHl, EeoRL, Pst, Sal, Smal, Sphl, and Xbal. Indicate the location of each restriction site in this sequence. (See Table 11.5 of restriction enzymes for their cleavage sites.)... 

Alzheimer s Disease. Figure 2 A(3 is derived from the APP by the sequential action of proteolytic activities exerted by (3- and y-secretases. APP-CTF is (C99) produced after cleavage of the APP by (3-secretase and represents the substrate of the y-secretase. The yellow box marks membrane embedded amino acid residues of A(3 peptide. Scissors represent the main cleavage sites of (3- and y-secretase, e.g. the e- and y-cleavages at positions 49,46, 42, 40 and 38. 

In about 30-40% of patients with suspected inherited thrombophilia the PC-pathway is disturbed by a mutation ofFV (FV-Leiden). TheFV-Leiden mutation affects one of the APC cleavage sites within the FV molecule. As a consequence, mutated FVa becomes resistant to rapid APC inactivation (APC resistance). About 4-7% of the middle European population cany this polymorphism of FV. Inborn deficiencies of Protein-S or Protein-C are much less frequent (< < 1% and 0.2-0.4%, respectively). 

The maturation of the precursor protein involves their proteolytic cleavage. There are two proteins important in this cleavage, so-called processing protease and protease enhancing peptide. These are now believed to be nonidentical subunits of the same enzyme. The structural requirements for recognition of the cleavage site are not fully understood and except for a positively charged residue at position (-2) there is no consensus sequence around this site. 

Crystallographic studies imply that although little sequence homology exists between the different protease cleavage sites, what is conserved is the shape that they adopt within the active site of the enzyme (Prabu-Jeyabalan et al. 2002). This shape has been termed the substrate envelope and represents the consensus volume overlapping the majority of the substrates. Most likely, HIV-1 protease recognizes a particular peptide sequence as being a substrate by a combination of accessibility and the shape the sequence can adopt. 

As an example of purification via the ELP fusion approach Meyer and Chilkoti (Fig. 9, left), purified the proteins thioredoxin and tendamistat. For this purpose these target proteins and ELP were genetically fused via a short peptide sequence that included a thrombin cleavage site, which allows the removal of the ELP tag after the purification is completed. The general outline of the purification procedure... 

Fig. 15 Amino acid sequences of artificial extracellular matrix (aECM) proteins. Each protein contains a TV tag, a histidine tag, a cleavage site, and elastin-like domains with lysine residues for crosslinking. The RGD cell-binding domain is found in aECM 1, whereas aECM 3 contains the CS5 cell-binding domain. aECM 2 and aECM 4 are the negative controls with scrambled binding domains for aECM 1 and aECM 3, respectively. Reprinted from [121] with permission from American Chemical Society, copyright 2004...

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