Abstract
The possibility of controlling interactions at interfaces
and surfaces of solid materials is highly interesting for
a wide range of nanotechnological applications. In
Nature, evolution has developed a wide range of proteins
and peptides that possess the ability to recognize,
specifically bind, and modify the surfaces of solid
materials. The natural evolution processes can be
mimicked in the laboratory scale with the use of a
directed evolution approach, for instance, based on the
selection of short material-specific peptides from
combinatorial libraries displayed on the surface of
bacteriophages or bacterial cells. Selected from billions
of different variants, material-specific peptides can be
studied to define their sequence, structure, binding
properties, and subsequently engineered to tailor their
function for practical applications.
In this work, a phage display was used to identify
peptides binding to diamond-like carbon (DLC). DLC is an
amorphous form of carbon, with chemical and physical
properties resembling natural diamond. It is used as a
coating material in many industrial and biomedical
applications. Peptides binding to DLC were selected from
the commercial phage display library (Ph.D.-12). During
the selection process phages displaying longer than the
expected 12-mer peptides (generally present in Ph.D.-12
library) were enriched. Binding studies by phage ELISA
and titer analysis indicated that enriched phages
displaying long (42-57-mer) peptides bind more
efficiently to DLC surface compared to the clones
displaying standard 12-mer sequences. Selected DLC
binding peptides (DLCBP) were fused to the alkaline
phosphate (AP), which was used as a reporter enzyme in
order to determinate their binding properties in a
different molecular context. The adsorption of the
DLCBP-AP fusions on DLC was quantified using the AP
enzymatic activity, and verified by ellipsometry. A
57-mer peptide, DLCBP11(L)-AP, showed the highest binding
to DLC with a binding Kd value of 63 nM. Studies of
different variants of the peptide demonstrated that its
shorter form (pep_L), composed of 29 amino acids, had
very similar binding properties. The structural basis of
the function of the pep_L peptide was investigated by
mutagenesis approach. The influence of mutations was
measured using a competition assay in which peptide
variants in free, soluble form competed for binding to
DLC with the
pep_L-AP fusion protein. Analysis of point mutations
demonstrated that a positive charge is important for
peptide function. Rearrangement of the order of amino
acid residues in the primary sequence showed that the
peptide's function is dependent on its chemical
composition and likely the overall three-dimensional
structure. Engineering of the peptide to different
multivalent forms showed that binding affinity and
kinetics of the multimers can be tailored by specific
structural design. Finally, it was demonstrated that the
peptides can be successfully utilized in
nanotechnological applications, i.e., for self-assembling
coatings on the DLC surface, and for controlling
properties of a colloidal form of DLC.
Besides finding and characterizing peptides binding to
DLC, the work also highlights various challenges of the
directed evolution techniques, for example, selection of
target unrelated peptides during biopanning, and the
necessity of multiple independent ways of analyzing the
functionality of selected peptides. Moreover, the most
frequent mistakes that are found in the literature when
analyzing results of the biopanning are discussed.
Original language | English |
---|---|
Qualification | Doctor Degree |
Awarding Institution |
|
Supervisors/Advisors |
|
Award date | 6 Mar 2015 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 978-951-38-8216-7 |
Electronic ISBNs | 978-951-38-8217-4 |
Publication status | Published - 2015 |
MoE publication type | G5 Doctoral dissertation (article) |
Keywords
- inorganic-binding peptides
- material specific peptides
- diamond-like carbon (DLC)
- phage display
- molecular recognition
- structure-function relationship
- multivalent peptides
- colloidal DLC