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INAUGURAL-DISSERTATION





zur Erlangung der Doktorwürde der
Naturwissenschaftlich-Mathematischen Gesamtfakultät
der Ruprecht-Karls-Universität Heidelberg








vorgelegt von
Dipl.-Chem. Patricia Maria Wolny
Geboren in Beuthen/Polen











Tag der mündlichen Prüfung: 02.03.2010







NOVEL MODEL SYSTEMS FOR THE INVESTIGATION OF
POLYVALENT PROTEIN-HYALURONAN INTERACTIONS ON
THE CELL SURFACE




















Gutachter/ Referees






Prof. Dr. J. P. Spatz Prof. Dr. Alain R. Brisson

Physikalisch-Chemisches-Institut Laboratoire d`Imagerie Moleculaire et
Universität Heidelberg Nano-Bio-Technologie
Max-Planck-Institut für Université Bordeaux 1
Metallforschung, Stuttgart





In den Wissenschaften ist viel Gewisses, sobald man sich von den
Ausnahmen nicht irre machen läßt und die Probleme zu ehren weiß.

Johann Wolfgang von Goethe, (1749 - 1832),
deutscher Dichter der Klassik, Naturwissenschaftler und Staatsmann




Für meine Eltern.
Für Johannes.

Abstract
The gel-like hyaluronan (HA) rich coat that surrounds many cells has been linked to a variety
of vital cellular functions, but the regulatory mechanisms at the cell-HA matrix interface
remain poorly understood. For the thorough investigation of specific interactions between the
cell surface and HA as well as the structural properties of this supra-molecular matrix, it is
desirable to switch from the complex cellular environment to simplified systems. This work
aims to find routes towards the development of new in-vitro model systems of HA-rich coats.
One approach relies on the immobilization of ectodomains of HA's main cell surface receptor,
CD44, on supported lipid membranes (SLBs). Model surfaces with tunable receptor density
are exploited to investigate the polyvalent interaction between HA and CD44 in a biologically
relevant arrangement. On surfaces that provide a high density of receptors, HA binding
increases in a sigmoidal fashion with molecular weight, and becomes reversible below
30 kDa. The physico-chemical properties of these HA films reveal many similarities between
the binding behavior of HA chains and flexible polymer chains adsorbing to a homogeneously
attractive surface. On surfaces with low receptor surface density binding of HA of sufficiently
high molecular weight is irreversible for all, and proportional to the amount of receptors.
Quantifying the number of receptors that are available per HA chain provides insight how
polyvalent interactions stabilize HA binding on receptor covered surfaces. HA binding is
though regulated by both polyvalency and the intrinsic affinity of individual receptors, and we
outline a method to disentangle the contributions of both effects.
Another approach envisages the immobilization of HA via a terminal biotin moiety at
controlled anchor separation. The binding and activity of biotin-receptors on gold and silica is
systematically examined with the objective to selectively functionalize gold nanostructured
surfaces. We find that streptavidin adsorbs in a functional conformation on gold while
resisting binding to silica. First attempts to exploit this selectivity on gold nanostructures are
presented.
The confinement of the model systems to solid supports enables qualitative and quantitative
characterization by complementary surface sensitive techniques. Here, quartz crystal
microbalance with dissipation monitoring and ellipsometry - alone or in combination - as well
as microinterferometry provide detailed insight into the formation, stability and morphology
of the model surfaces and the HA films, and involved molecular interactions.
The developed model systems with tunable properties can serve as a well-controlled
experimental platform for the investigation of the interactions between proteins and HA in a
supra-molecular context, and between cells and HA-matrices.

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