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The molecular recognition of phosphorylated proteins by designed polypeptides conjugated to a small molecule that binds phosphate
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Lars Baltzer)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Lars Baltzer)
2011 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, no 22, 7697-7704 p.Article in journal (Refereed) Published
Abstract [en]

The conjugation of polypeptides from a designed set to the small molecule ligand 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, which in the presence of Zn2+ ions binds inorganic phosphate, has been shown to provide a polypeptide conjugate that binds α-casein, a multiply phosphorylated protein, with a dissociation constant KD of 17 nM. The measured affinity is more than three orders of magnitude higher than that of the small molecule ligand for phosphate and the binding of 500 nM of α-casein was not inhibited by 10 mM phosphate buffer, providing a 2000-fold excess of phosphate ion over protein. The selectivity for phosphoproteins was demonstrated by extraction of α-casein from solutions of various complexity, including milk and human serum spiked with α-casein. In addition to α-casein, β-casein was also recognized but not ovoalbumin. Conjugation of a polypeptide to the zinc chelating ligand was therefore shown to give rise to dramatically increased affinity and also increased selectivity. A set of polypeptide conjugates is expected to be able to capture a large number of phosphorylated proteins, perhaps all, and in combination with electrophoresis or mass spectrometry become a powerful tool for the monitoring of phosphorylation levels. The presented binder can easily be attached to various types of surfaces; here demonstrated for the case of polystyrene particles. The example of phosphoproteins was selected since posttranslational phosphorylation is of fundamental importance in cell biology due to its role in signaling and therefore of great interest in drug development. The reported concept for binder development is, however, quite general and high-affinity binders can conveniently be developed for a variety of proteins including those with posttranslational modifications for which small molecule recognition elements are available.

Place, publisher, year, edition, pages
2011. Vol. 9, no 22, 7697-7704 p.
Keyword [en]
Molecular recognition, binders, peptide - small molecule conjugate, conjugates
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Bioorganic Chemistry; Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-161010DOI: 10.1039/c1ob06154bISI: 000296203700017OAI: oai:DiVA.org:uu-161010DiVA: diva2:454051
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Peptide Conjugates as Useful Molecular Tools
Open this publication in new window or tab >>Peptide Conjugates as Useful Molecular Tools
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The conjugation of a small organic molecule to synthetic polypeptides from a designed set has been shown to give rise to binders with high affinity and selectivity for the phosphorylated model proteins α-casein and β-casein but not for ovoalbumin. The small organic molecule that was used for this purpose is comprised of two di-(2-picolyl)amine groups assembled on a dimethylphenyl scaffold, and is capable of complexing two Zn2+ ions to form chelates that bind the phosphate ion. The designed polypeptides used for binder construction have no precedence in nature and do not show any prior selectivity favouring any single protein. The polypeptide conjugate binders showed high affinity towards the model protein α-casein, the binder molecule 4C15L8-PP1 bound α-casein with a dissociation constant KD of 17 nM, although the di-(2-picolyl) amine based chelate in the presence of Zn2+ bound phosphate ion with dissociation constants in the low mM range. The observed affinity is due to interactions between the Zn2+ chelate and the phosphate groups of α-casein and also to interactions between the polypeptide scaffold and α-casein. The binder was found to selectively extract α-casein from buffer, bovine milk and human serum spiked with α-casein. The flexible construction of the binder permits for flexible modifications like attachment of fluorophores for titrations and quantifications. The binders were shown to efficiently capture α-casein from human serum when immobilized on solid support in a continuous flow system and to release the captured α-casein upon a simple change of pH using 0.1% acqueous trifluoroacetica acid. The developed technology brings new opportunities in investigating posttranslational phosphorylation events that are involved in signaling cascades and triggering many biologically relevant functions.

A new chemical linker technology has also been developed for the purpose of conjugating biomolecules taking advantage of amino groups for the conjugation. By combining two esters with different reactivities, separated by an aliphatic chain, a molecular tool was developed that allows for controlled conjugation of biomolecules. The two esters react at different rates and can therefore be separated and allowed to react under different conditions in each step, thereby allowing for selective linkage formation between the subunits. The size of the spacer can be varied by selecting the appropriate dicarboxylic acid. The developed technology was shown to provide specificity in heteroconjugate formation in the assembly of a variety of heteroconjugates where polypeptides were combined with other peptides, carbohydrates and proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 883
Keyword
peptide conjugates, designed peptides, polypeptides, molecular recognition, linker, linking technology, bioconjugation, conjugation, conjugates
National Category
Organic Chemistry
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-161013 (URN)978-91-554-8227-5 (ISBN)
Public defence
2011-12-15, B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-11-25 Created: 2011-11-04 Last updated: 2012-01-03Bibliographically approved

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Ślósarczyk, Adam T.Baltzer, Lars

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