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α2A-adrenergic receptor derived peptide adsorbates: a g-protein interaction study
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
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2006 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 22, no 17, 7260-7264 p.Article in journal (Refereed) Published
Abstract [en]

The affinity of α2A-adrenergic receptor (α2A-AR) derived peptide adsorbates for the functional bovine brain G-protein is studied in the search for the minimum sequence recognition. Three short peptides (GPR-i2c, GPR-i3n, and GPR-i3c) are designed to mimic the second and third intracellular loops of the receptor. X-ray photoelectron spectroscopy is used to study the chemical composition of the peptides and the binding strength to the surfaces. Chemisorption of the peptides to the gold substrates is observed. Infrared spectroscopy is used to study the characteristic absorption bands of the peptides. The presence of peptides on the surfaces is verified by prominent amide I and amide II bands. The interaction between the peptides and the G-protein is studied with surface plasmon resonance. It is shown that GPR-i3n has the highest affinity for the G-protein. Equilibrium analysis of the binding shows that the G-protein keeps its native conformation when interacting with GPR-i3c, but during the interaction with GPR-i2c and GPR-i3n the conformation of G-protein is changed, leading to the formation of aggregates and/or multilayers.

Place, publisher, year, edition, pages
2006. Vol. 22, no 17, 7260-7264 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:liu:diva-50157DOI: 10.1021/la052801rOAI: oai:DiVA.org:liu-50157DiVA: diva2:271053
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically 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)978–91–7393–050–5 (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: 2015-06-03Bibliographically approved
2. Bioactive adsorbates on gold surfaces: structural properties and bio-interaction studies
Open this publication in new window or tab >>Bioactive adsorbates on gold surfaces: structural properties and bio-interaction studies
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis investigates the properties of biomolecular, model adsorbates on gold such as amino acid derivatives, peptides and related organic molecules. Subsequent bin-interaction studies were also conducted. The physico-chemical and structural properties of the adsorbates were characterized using X-ray Photoelectron Spectroscopy (XPS), Infrared-Reflection Absmption Spectroscopy (IRAS) and Near-Edge X-ray Absmption Fine Structures spectroscopy (NEXAFS). Complementary techniques such as Null ellipsometry and Cyclic Voltammetry (CV) were also used. The interaction of the bioactive monolayers with biologically relevant molecules, such as proteins and metal ions, were investigated using Surface Plasmon Resonance (SPR) spectroscopy and Electrochendcallmpedance Spectroscopy (EIS).

The first part of the thesis is directed towards the interaction of bovine-brain G-protein with adsorbates involving arginine residues and receptor-derived peptides mimicking the 2nd and 3rd intracellular (ic) loop of the α2A Adrenergic G-protein coupledreceptor (GPCR). The general aim is to find a peptide sequence that will selectively, with high affinity, interact with the G-protein. The specific aims were to examine the importance of the presence of positively charged arginine residues, to investigate the influence of molecular orientation of the adsorbates, and to verify which intracellular loop has the highest affinity to the G-protein. The investigation involved characterizing the chemical composition and the molecular orientation of Arginine-based dipeptide adsorbates (Arg-Cys and Arg-Cysteandne) and receptor-detived peptides (GPR1R also labeled GPRi3c, GPR1K, GPR1A, GPRi2c, GPRi3n) innnobilized on gold surfaces, followed by G~protein interaction studies. On all the adsorbates subjected to interact with G-proteins, the presence of arginine residues was proven to be of special importance in the affinity of G-proteins. A molecularly"oriented Arg-Cysteamine, with main molecular axis perpendicular to the gold surface, showing more available arginines, attracts more G-proteins as compared to Arg-Cys that has a compact configuration when adsorbed on gold. The peptide adsorbates derived from the third ic loop (GPRi3c and GPRi3n) have higher affinity than peptides derived from the second ic loop (GPRi2c). This shows that this arginine-rich area of the third ic loop has a major influence on the affinity and selectivity of G-proteins.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2005. 76 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 988
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-30969 (URN)16648 (Local ID)91-8545-775-2 (ISBN)16648 (Archive number)16648 (OAI)
Public defence
2005-12-16, Hörsal Plank, Fysikhuset, Campus Valla, Linköping, 10:15 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-11-23

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Vahlberg, CeciliaPetoral, Rodrigo JrLindell, CharlottaBroo, KerstinUvdal, Kajsa

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