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Orientation and Conformation of Single Macromolecules on Unmodified and Functionalized Surfaces
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Surface Biotechnology.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis methods for investigation of orientation and conformation of individual macromolecules on surfaces are presented as well as novel methods for functionalization of silicon chips with the possibility to get an ordered immobilization of antibodies.

Two novel methods are presented which makes it possible to investigate the orientation of individual macromolecules on different kinds of surfaces with AFM. One is based on threshold patterning where, depending on substrate, side- and end-on adsorbed immunoglobulin molecules could be detected. The other method is using the principle of site-specific ligands where the orientation of proteins adsorbed to various surfaces was evaluated. By measuring the increase in protein volume of the formed protein-ligand complexes with AFM, the amount of protein having an orientation that allows binding can be estimated.

The influence of surface chemistry on protein structure was examined using human serum fibronectin adsorbed to hydrophilic and hydrophobic surfaces, where a major difference in structure were seen depending on surface properties.

In addition, methods for surface functionalization have been developed which are suitable for immobilization of macromolecules and for basic studies of macromolecule/surface interactions at the nanometer scale. In an effort to immobilize protein in a specific orientation that could be studied with AFM, a new method for preparing reactive disulfides based upon a mixed reaction with 2,2’-dipyridyldisulfide and 2-thiopyridone to a mercapto-silanized silica surface was presented. The possibility to covalently bind proteins to this surface was examined, using beta-galactosidase and Fab’-fragments of IgG.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2002. , 62 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 694
Keyword [en]
Biotechnology, Atomic Force Microscopy, protein orientation, protein adsorption, surface derivatization
Keyword [sv]
Bioteknik
National Category
Industrial Biotechnology
Research subject
Surface Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-1854ISBN: 91-554-5259-0 (print)OAI: oai:DiVA.org:uu-1854DiVA: diva2:161418
Public defence
2002-04-11, B41, Uppsala, 14:00
Opponent
Available from: 2002-03-19 Created: 2002-03-19Bibliographically approved
List of papers
1. TM-AFM Threshold Analysis of Macromolecular Orientation: A Study of the Orientation of IgG and IgE on Mica Surfaces
Open this publication in new window or tab >>TM-AFM Threshold Analysis of Macromolecular Orientation: A Study of the Orientation of IgG and IgE on Mica Surfaces
1998 (English)In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 206, no 2, 475-481 p.Article in journal (Refereed) Published
Abstract [en]

Adsorption and orientation properties of two different types of immunoglobulin molecules on derivatized and native mica surfaces were investigated using TM-AFM. The analyses included height measurements at two different pH values and a new technique, presented here as threshold analysis, which displays the outer mantle shape of an adsorbed protein. A major difference in preferential orientation is observed upon comparing the adsorption of the two proteins onto the different surfaces. The characteristics of both the adsorbed immunoglobulin and the surface are important for any preferential orientation of the adsorbed protein.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89709 (URN)10.1006/jcis.1998.5630 (DOI)9756659 (Scopus ID)
Available from: 2002-03-19 Created: 2002-03-19 Last updated: 2013-05-30Bibliographically approved
2. A method for studying protein orientation with atomic force microscopy using relative protein volumes
Open this publication in new window or tab >>A method for studying protein orientation with atomic force microscopy using relative protein volumes
2001 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 105, no 10, 2062-2069 p.Article in journal (Refereed) Published
Abstract [en]

A method for studying protein orientation is described, in which the relative volumes of single proteins and single molecule complexes are measured using atomic force microscopy (AFM). Site-specific ligands are used as “probes” to bind to surface adsorbed proteins. The quantity of formed complexes gives an estimate of the amount of protein oriented in such a way as to allow ligand binding. The volume distribution for single proteins adsorbed to a surface was calculated and fitted to a Gaussian function. This volume distribution was used to localize the same proteins on surfaces with protein−ligand complexes, thus rendering it possible to find the amount of complex formed. Two model systems were used: one with two different mouse monoclonal antibodies of IgG1 type (mAb's against human serum transferrin, hST) adsorbed on silicon surfaces, and one with hST adsorbed to unmodified mica and aminated mica. The adsorbed proteins were allowed to react with a site-specific ligand, which binds to a defined region of the adsorbed protein (hST in the case of adsorbed mAb and lectin in the case of adsorbed hST). A great difference in ligand binding was found between the two antibodies adsorbed to the same type of surface as well as between hST adsorbed to different surfaces. This differance can be attributed to different orientation of the proteins on the surface. The general approach of this method suggests that it can be used for almost any site-specific molecule, either for surface orientation studies or studies where single molecule interactions need to be investigated.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89710 (URN)10.1021/jp003957x (DOI)
Available from: 2002-03-19 Created: 2002-03-19 Last updated: 2013-06-12Bibliographically approved
3. Surface dependant conformations of human plasma fibronectin adsorbed to silica, mica and hydrophobic surfaces, studied with Atomic Force Microscopy
Open this publication in new window or tab >>Surface dependant conformations of human plasma fibronectin adsorbed to silica, mica and hydrophobic surfaces, studied with Atomic Force Microscopy
In: J. Biomed. Mater. Res.Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-89711 (URN)
Available from: 2002-03-19 Created: 2002-03-19Bibliographically approved
4. A Novel Method for Preparation of Disulfides on Silicon
Open this publication in new window or tab >>A Novel Method for Preparation of Disulfides on Silicon
Show others...
2001 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 17, no 20, 6056-6058 p.Article in journal (Refereed) Published
Abstract [en]

This work describes an efficient novel method to incorporate reactive disulfide bonds onto a silica surface under mild reaction conditions. The reactive thiol groups introduced onto the silicon surface in the first reaction step will be oxidized but easily converted into highly reactive thiopyridyl groups, which can therefore easily be utilized for further organic synthesis involving thiol-containing molecules. This is done in a way that yields approximately a monolayer of reactant on the surface, thereby not adding to the roughness of the surface, of special importance, for instance, for single molecule interaction studies.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89712 (URN)10.1021/la0155092 (DOI)
Available from: 2002-03-19 Created: 2002-03-19 Last updated: 2013-06-12Bibliographically approved
5. Atomic Force Microscopy studies of proteins immobilized on silica by thiol-disulfide exchange
Open this publication in new window or tab >>Atomic Force Microscopy studies of proteins immobilized on silica by thiol-disulfide exchange
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-89713 (URN)
Available from: 2002-03-19 Created: 2002-03-19 Last updated: 2010-01-13Bibliographically approved

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