Abstract
BackgroundTrypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction.ResultsBased on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme.ConclusionThe variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0109-5) contains supplementary material, which is available to authorized users.
Highlights
Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues
The human enzyme trypsinogen shows a distinct distribution of protein surface charges and may be an attractive model protein to study the influence of electrostatic forces
We describe the modification of human trypsinogen by the exchange of several amino acid residues on the protein surface and its effect on specific properties of the protein such as the protein–protein interaction behavior of trypsinogen with enteropeptidase and active trypsin
Summary
Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. Human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen can be autoactivated without the involvement of enteropeptidase This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and is based on a protein–protein interaction. The human enzyme trypsinogen shows a distinct distribution of protein surface charges and may be an attractive model protein to study the influence of electrostatic forces. This trypsinogen autoactivation process is mostly based on the fact that active trypsin can activate trypsinogen by cleaving off its activation peptide in a similar way as occurs with the activation enzyme enteropeptidase. As the same amount and type of trypsin was used throughout these studies, this ensures that autoactivation was characterized in a comparable way
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