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Structure analysis and molecular recognition studies of bio-functionalized surfaces
Linköping University, Department of Physics, Chemistry and Biology, Molecular Surface Physics and Nano Science. Linköping University, The Institute of Technology.
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: urn:nbn:se:liu:diva-71457ISBN: 9789173930505 (print)OAI: oai:DiVA.org:liu-71457DiVA: diva2:449093
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
List of papers
1. α2A-adrenergic receptor derived peptide adsorbates: a g-protein interaction study
Open this publication in new window or tab >>α2A-adrenergic receptor derived peptide adsorbates: a g-protein interaction study
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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.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-50157 (URN)10.1021/la052801r (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved
2. Mixed monolayers to promote G-protein adsorption: α2A- Adrenergic receptor-derived peptides coadsorbed with formyl-terminated oligopeptides
Open this publication in new window or tab >>Mixed monolayers to promote G-protein adsorption: α2A- Adrenergic receptor-derived peptides coadsorbed with formyl-terminated oligopeptides
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.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-47944 (URN)10.1021/la063447f (DOI)000248229900028 ()
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
3. Noradrenaline and a Thiol Analogue on Gold Surfaces: An Infrared Reflection-Absorption Spectroscopy, X-ray Photoelectron Spectroscopy, and Near-Edge X-ray Absorption Fine Structure Spectroscopy Study
Open this publication in new window or tab >>Noradrenaline and a Thiol Analogue on Gold Surfaces: An Infrared Reflection-Absorption Spectroscopy, X-ray Photoelectron Spectroscopy, and Near-Edge X-ray Absorption Fine Structure Spectroscopy Study
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2011 (English)In: The Journal of Physical Chemistry C, ISSN 1932-7447, E-ISSN 1932-7455, Vol. 115, no 1, 165-175 p.Article in journal (Refereed) Published
Abstract [en]

Self-assembled monolayers and multilayers of a noradrenaline analogue (Nor-Pt) on gold substrates as well as multilayers of noradrenaline have been investigated by means of the molecular orientation, the molecule surface interaction, the molecular composition and the functional group availability for further biointeraction processes, using X-ray photoelectron spectroscopy (XPS), infrared reflection absorption spectroscopy (IRAS), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. A chemical shift (1.7 eV) of the S 2p peak to lower binding energies is observed, in the XPS spectrum, indicating that the Nor-Pt molecules are chemisorbed onto the gold substrate. The IR results show that Nor-Pt adsorbate has the C=O stretching vibration modes parallel oriented relative to the gold substrate. The average tilt angle of the aromatic ring relative to the gold surface normal is determined to be approximately 51 degrees, based on the NEXAFS measurements on Nor-Pt monolayers. The experimental results and assignments are supported with theoretical studies where we use the building block principle in the spectral analysis and compare with the measurements of noradrenaline and Nor-Pt. The theoretical calculations are shown to be useful; for angle dependence NEXAFS studies as resonances with fully pi* or sigma* character are preferred for correct analysis.

Place, publisher, year, edition, pages
American Chemical Society, 2011
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-66293 (URN)10.1021/jp105696j (DOI)000285818200024 ()
Available from: 2011-03-11 Created: 2011-03-11 Last updated: 2017-12-11Bibliographically approved
4. Phenylboronic ester- and Phenylboronic acid-terminated alkanethiols on Gold Surfaces
Open this publication in new window or tab >>Phenylboronic ester- and Phenylboronic acid-terminated alkanethiols on Gold Surfaces
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In this work, it is shown that well-organized monolayer of phenylboronic ester-terminated thiol (BOR-Capped) on gold surfaces can be prepared. Our results also show that the BORCapped molecular system can be cleaved directly on the surface, resulting in an unprotected BOR-Uncapped monolayer with the boronic acid functional groups available for dopamine coordination. The monolayers of BOR-Capped and BOR-Uncapped were characterized using infrared spectroscopy, near edge X-ray absorption fine structure spectroscopy, X-ray photoelectron spectroscopy, ellipsometry and contact angle goniometry. The X-ray photoelectron spectroscopy results showed that both BOR-Capped and BOR-Uncapped are chemically linked to the gold substrate. According to the infrared spectroscopy results, the main component of the C=O vibrational mode present in the amide moiety is perpendicular oriented relative to the gold surface normal for the BOR-Capped molecular system. The near edge X-ray absorption fine structure spectroscopy resonance peak located at approximately 285 eV, assigned to π* transitions, was used to estimate the average tilt angle of the vector parallel to the π* orbitals of the aromatic ring relative to the gold surface normal. The average tilt angle is estimated to be approximately 63º for BOR-Capped monolayer on gold surfaces. The aromatic ring of the BOR-Uncapped molecule has a more tilted orientation compared to BOR-Capped. The experimental infrared spectroscopy and near edge X-ray absorption fine structure spectroscopy results were supported with theoretical modeling including calculations of vibrational modes and of excitation processes.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-71455 (URN)
Available from: 2011-10-19 Created: 2011-10-19 Last updated: 2011-10-19Bibliographically approved
5. The Structure of Benzenesulfonamide-Terminated Thiol on Gold Surfaces and the Interaction with Carbonic Anhydrase
Open this publication in new window or tab >>The Structure of Benzenesulfonamide-Terminated Thiol on Gold Surfaces and the Interaction with Carbonic Anhydrase
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

A well-structured and robust biomolecular monolayer based upon a benzenesulfonamideterminated alkane thiol, to be used as a model system for molecular recognition processes, was prepared. The benzenesulfonamide-terminated thiol adsorbed onto gold substrates was characterized using X-ray photoelectron spectroscopy, near edge X-ray absorption fine structure spectroscopy, infrared-reflection absorption spectroscopy and ellipsometry. The results showed that the benzenesulfonamide-terminated alkane thiol forms a wellorganized molecular layer on the gold substrates. The orientation of the aromatic ring relative to the gold surface was investigated by means of the angle defined as the normal to the aromatic ring relative to the normal to the gold surface. It was shown that the average tilt angle is approximately 62º. In a second step, the  benzenesulfonamideterminated thiol monolayer was exposed to carbonic anhydrase, which is an enzyme and a therapeutic target. Benzenesulfonamides are used in biomedical applications as inhibitors for carbonic anhydrase. Our purpose in this study was to investigate the recognition capability of the benzenesulfonamide when designed as a thiol monolayer. The interaction between the benzenesulfonamide-terminated monolayer and carbonic anhydrase was studied using ellipsometry and surface plasmon resonance. The results show that the benzenesulfonamide-terminated thiol adsorbed onto the gold substrates is able to bind carbonic anhydrase. The results also indicate that the interaction is specific.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-71456 (URN)
Available from: 2011-10-19 Created: 2011-10-19 Last updated: 2011-10-19Bibliographically approved

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