The importance of correct protein concentration for kinetics and affinity determination in structure-function analysis.
In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A(280). After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (k(a)) and affinity (K(D)), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.
GE Healthcare Bio-Sciences AB. Ewa.Pol@ge.com
Ewa
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Pol
D000818:Animals; D002417:Cattle; D055313:Cystatin B / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010206:Papain / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance / Q000379:methods
D055313:Cystatin B; D010206:Papain