Time course of proteolysis

Figure 3. Time course of proteolysis of ngCenA and ngCex. 300 /zg of ng CenA (A) or ngCex (B) dissolved in 725 /zl of phosphate buffer containing...

Once the most useful, in terms of ability to preferentially cleave either the apo or the holo form of the binding protein, protease(s) has (have) been identified, It is necessary to perform a time course of proteolysis. This aids in both the determination of the order of evolution of cleavage products and in the optimal reaction time for production of the desired proteolytic fragment. The time-course also provides additional information on the relative rates of proteolysis of the apo and holo forms of each of the binding proteins. 

The proteolysis is done as in the time-course of proteolysis, except that... 

Using the results from the time-course of proteolysis experiment, Subheading 3.3., determine the optimal reaction time in order to obtain the maximal amount of the desired fragment. 

Figure 9. Time course of thermal destabilization of chicken ovomucoid (OM) by treatment (proteolysis) with proteinase K (PrK).

The highly successful extension of site-directed suppression mutagenesis to a Xenopus oocyte system by the Dougherty and Lester labs discussed above has permitted structural, functional and kinetic studies of ion channels in intact cells [30-32, 62]. Recently, the introduction of residues that allow photochemical proteolysis and photo deprotection of protein side chains has been applied to the study of ion channels. Miller et al. introduced o-nitrobenzyl-pro-tected tyrosines at three positions in the a-subunit of nAChR [62 c]. The o-nitro-benzyl group was then removed photolytically to produce the wild-type residue during the course of a voltage-clamp study, so that time-resolved measurements could be made. 

An offshoot of this protocol is the limited proteolysis strategy, in which proteolysis is restricted to a single event that cleaves the complex into two complementary fragments only this identifies the most susceptible bond in the protein and hence the protease-accessible amino acid residue. To identify other accessible residues, proteolysis is performed in a time-course fashion with a suite of enzymes of diverse specificities. The potential of the limited proteolysis strategy has been demonstrated by mapping the topology of Ca2+-calmodulin-melittin ternary complex.165... 

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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