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Mixed monolayers to promote G-protein adsorption: α2A- Adrenergic receptor-derived peptides coadsorbed with formyl-terminated oligopeptides
Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, Faculty of Science & Engineering.
2007 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, no 16, 8474-8479 p.Article in journal (Refereed) Published
Abstract [en]

Pure and mixed monolayers of a synthetic peptide, GPR-i3n, derived from the third intracellular loop of the α2 adrenergic receptor and a shorter inactive oligopeptide, N-formyl-(Gly)3-(Cys) (called 3GC), were prepared on gold surfaces. The mixing ratio of the GPR-i3n and 3GC was used to control G-protein binding capability. The GPR-i3n peptide is specially designed for bovine G-protein selectivity and has been proven to have high affinity to G-proteins [Vahlberg, C.; Petoral, R. M., Jr.; Lindell, C.; Broo, K.; Uvdal, K. Langmuir 2006, 22 (17), 7260−7264]. Pure 3GC monolayers show very low protein adsorption capability. In this study, 3GC is chosen as a coadsorbent, with the aim to induce molecular conformational changes during monolayer formation to enhance G-protein adsorption. A full characterization of the mixed monolayers was done. The monolayer thickness and the mass-related surface coverage for both GPR-i3n and 3GC were investigated using radio labeling. The GPR-i3n was labeled by 125I-targeting tyrosine, and the activity was measured by using radioimmunoassay (RIA). The formation and chemical composition of GPR-i3n and 3GC monolayers were investigated using X-ray photoelectron spectroscopy, and it is shown that both GPR-i3n and 3GC bind chemically to the gold surface. The interaction between the mixed monolayers and G-proteins was investigated by means of real-time surface plasmon resonance. There is a higher protein binding capacity to the monolayer when the GPR-i3n peptide is intermixed with the 3GC coadsorbent, despite the fact that the 3GC itself has a very low G-protein binding capability. This supports a molecular reorientation at the surface, while 3GC is intermixed with GPR-i3n.

Place, publisher, year, edition, pages
2007. Vol. 23, no 16, 8474-8479 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:liu:diva-47944DOI: 10.1021/la063447fISI: 000248229900028OAI: oai:DiVA.org:liu-47944DiVA: diva2:268840
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Structure analysis and molecular recognition studies of bio-functionalized surfaces
Open this publication in new window or tab >>Structure analysis and molecular recognition studies of bio-functionalized surfaces
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological and chemical reactions involved in physiological processes are often complex and very sophisticated. Such processes can be mimicked in the laboratory to obtain important knowledge, valuable for the  development of new diagnostic methods, drugs and biosensors. This  thesis includes investigations of bio-functionalized surfaces that can be used as model systems to mimic naturally existing biomolecular recognition processes.

In this thesis, three different peptides, of our own design, that mimic parts of the second and third intracellular loops of the α2A-adrenergic receptor, are studied. The peptides were immobilized onto gold substrates, through thiol chemistry. The interaction between the peptides and the G-protein was investigated using surface plasmon resonance (SPR). The G-protein showed the highest binding capability for surfaces functionalized with a peptide mimicking the n-terminal of the third intracellular loop (GPR-i3n). The binding was enhanced when the pure GPR-i3n peptide was mixed with a short oligopeptide (3GC). A tentative explanation for the obtained results is that the presence of the 3GC molecule enables conformational changes of the GPR-i3n monolayer which affect the interaction with the G-protein. The results from the SPR measurements also indicated that the conformation of the G-protein was kept intact during the interaction with a peptide mimicking the c-terminal of the third intracellular loop (GPR-i3c). Multilayers were formed on the surfaces functionalized with a peptide mimicking the second intracellular loop (GPR-i2c) and the GPR-i3n peptide. We suggest that conformational changes of the G-protein are induced during the interaction with the surfaces functionalized with the GPR-i3n and GPR-i2c peptides.

Comprehensive surface characterizations of four biomolecular systems, based upon the functional groups: noradrenaline, phenylboronic-ester, phenylboronic-acid and benzenesulfonamide, are presented in the thesis. The aim is to develop a platform for detailed molecular recognition studies on surfaces. The molecular systems were characterized using infrared spectroscopy, X-ray photoelectron spectroscopy, near edge X-ray absorption fine structure spectroscopy, ellipsometry and contact angle goniometry. Noradrenaline was chosen as it is a neurotransmitter that interacts with the extracellular loops of adrenergic receptors. In this work, the noradrenaline analogue (Nor-Pt) of our own design, was equipped with a -SH handle to be linked to surfaces and with the free noradrenaline group available for interaction studies. The Nor-Pt molecules were organized on the surfaces with the sulfur atom close to the gold substrate and the aromatic ring available for possible interactions with other biomolecules in the ambient media. The main component of the C=O vibrational mode present in the amide moiety had a parallel orientation relative to the plane of the gold surface, based on the infrared spectroscopy results. The phenylboronic system was designed as a simple mimicry of an  adrenergic receptor as the boronic acid functional group binds to diol containing molecules such as noradrenaline. The boronic  esterterminated alkane thiol (BOR-Capped) was chemisorbed onto gold substrates. We showed that BOR-Capped was linked to the gold substrate via thiolate bond formation and formed a well-organized monolayer. The pinacolyl protection group was removed directly from the BOR-Capped monolayer on the surfaces, which resulted in an unprotected monolayer terminated with the boronic acid functional group (BOR-Uncapped). The strong chemical bond to the gold substrate was retained during the deprotection procedure as only thiolate sulfur species were observed for the BOR-Uncapped molecular system. The benzenesulfonamide based molecule was designed as a model system for bioselective surfaces. An amine-terminated alkane thiol was adsorbed onto a gold substrate. In a second step, a benzenesulfonamide derivative was linked to the amine-terminated monolayer by the formation of an amide bond. We showed that the resulting benzenesulfonamide-terminated alkane thiol (AUT-C6) formed a well-organized and semi-thick monolayer on the gold substrate. The polarization dependence of NEXAFS was used to determine the average tilt angle of the aromatic ring structures of Nor-Pt, BOR-Capped, BOR-Uncapped and AUT-C6. The results indicate that the aromatic ring planes of BOR-Capped and AUT-C6 have a preferential orientation toward the surface normal. The aromatic ring structures of Nor-Pt and BOR-Uncapped were determined to have a more tilted orientation relative to the gold surface normal.

Finally, the interaction between carbonic anhydrase and the AUT-C6 molecule was investigated using surface plasmon resonance and ellipsometry. The surface immobilized benzenesulfonamide was shown to bind to carbonic anhydrase and the results indicated that the interaction is specific.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. 56 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1404
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-71457 (URN)9789173930505 (ISBN)
Public defence
2011-11-11, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2011-10-19 Created: 2011-10-19 Last updated: 2017-12-15Bibliographically approved
2. Bioactive monolayers and thin films for biomedical applications
Open this publication in new window or tab >>Bioactive monolayers and thin films for biomedical applications
2006 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis includes two projects. The first one is a study of organic molecular self assembled monolayers designed to promote selective protein adsorption processes. The second one describes a high quality, freestanding films based on biodegradable chitosan obtained through a controlled preparation process and investigated as a promising system for many applications including biomaterials.

Within the first part of this work it was explored selective adsorbates for molecular recognition based on positively charged peptide and it has studied how the biomolecules are expressed when adsorbed to the surface. The design and the preparation of a peptide modified surface were done to investigate G-protein interactions. Pure and mixed monolayers of a synthetic peptide, GPR-i3n, derived from the 3rd intracellular loop of the α2 Adrenergic Receptor and a smaller inactive oligopeptide, N-formyi-(Gly) 3- (Cys) called 3GC, were prepared. In this study, 3GC is chosen as a co-adsorbent, with the aim to induce molecular conformational changes of GPR-i3n during the monolayer formation to improve the G-protein adsorption to the functionalized surface.

The formation and the chemical composition of the peptide monolayers on the surface were investigated as well as the monolayers thickness and the mass related surface coverage. The interaction between the mixed monolayers and G-proteins was investigated by means of real time Surface Plasmon Resonance (SPR). There is a higher protein binding capacity to the monolayer when the GPR-i3n peptide is intermixed with the 3GC coadsorbent, despite the fact that the 3GC itself has a very low G-protein binding capability. This supports our theory that a molecular reorientation of the GPR-i3n peptide occurs on the surface when 3GC is intermixed with GPR-i3n.

The formation of SAMs is described in chapter 4 and a full characterization of the mixed monolayers is reported as well as a G-protein interaction study.

In terms of biomaterials, a fundamental understanding of the structure and the composition of the surfaces is necessary for a successful design or when interacting with human body.

The objective of the second study was to thoroughly investigate the properties of free standing chitosan films, prepared by dry phase inversion from chitosan solutions in acetic acid. The films of chitosan were transparent with very good flexibility, while thicker films were fragile, showing an increase of internal tension with thickness. Structural analysis by X-ray diffraction (XRD) proved that the films as initially prepared are almost amorphous. Subsequent annealing converted the amorphous films into a mixture of amorphous and crystalline phases. The investigations by thermogravimetry (TG), the derivative (DTG) and the differential thermal curves (DTA) showed that the thermal degradation of chitosan films as initially prepared proceeds in two stages. The first decomposition stage corresponds to a complex series of processes including dehydration of saccharide rings, depolymerization and decomposition of the acetylated and deacetylated units of the polymers.

The methods used for surface analysis can provide information about the biomaterials, information that can be used to ensure the surface reproducibility and the fundamental aspects toward the interaction of biological systems with living systems.

Place, publisher, year, edition, pages
Linköping: Linköping University, 2006. 42 p.
Series
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1268
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-36128 (URN)30009 (Local ID)91-85643-92-0 (ISBN)30009 (Archive number)30009 (OAI)
Note

Licentiate thesis no 1268

Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2013-12-17

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Savitchi, LuminitaVahlberg, CeciliaPetoral, Rodrigo JrUvdal, Kajsa

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