Diamond-like carbon binding peptides: Evolutionary selection, characterization, and engineering: Dissertation

Bartosz Gabryelczyk

Research output: ThesisDissertationCollection of Articles


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 languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
  • Linder, Markus, Supervisor, External person
Award date6 Mar 2015
Place of PublicationEspoo
Print ISBNs978-951-38-8216-7
Electronic ISBNs978-951-38-8217-4
Publication statusPublished - 2015
MoE publication typeG5 Doctoral dissertation (article)


  • inorganic-binding peptides
  • material specific peptides
  • diamond-like carbon (DLC)
  • phage display
  • molecular recognition
  • structure-function relationship
  • multivalent peptides
  • colloidal DLC


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