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Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2009 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 391, no 1, 39-44 p.Article in journal (Refereed) Published
Abstract [en]

The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K-D = 0.03 mM and a low-affinity site with K-D = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K-D = 5 mu M at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and Suggest that Ca2+ is an efficient regulator under physiological conditions.

Place, publisher, year, edition, pages
2009. Vol. 391, no 1, 39-44 p.
Keyword [en]
C-reactive protein (CRP); Calcium binding; SPR biosensor, Conformational change, pH dependency, Phosphocholine
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-128986DOI: 10.1016/j.ab.2009.04.037ISI: 000267063200006PubMedID: 19435596OAI: oai:DiVA.org:uu-128986DiVA: diva2:332397
Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2013-02-11Bibliographically approved
In thesis
1. Protein Interaction Studies with Low Molecular Weight Ligands: Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
Open this publication in new window or tab >>Protein Interaction Studies with Low Molecular Weight Ligands: Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays.   

For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions.   

The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings.

The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the ß3 GABAA receptor has the potential to become a standard method for handling membrane proteins.  

The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes.

The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 34 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1007
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-188328 (URN)978-91-554-8566-5 (ISBN)
Public defence
2013-02-14, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Available from: 2013-01-24 Created: 2012-12-14 Last updated: 2013-04-02Bibliographically approved

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