Peptides and proteins in dendritic assemblies

Multiple, simultaneous interactions are often used in biology to enhance the affinity and specificity of binding, an effect referred to as multivalency. This multivalency can be mimicked by anchoring multiple peptides and proteins onto synthetic dendritic scaffolds. The aim of this research was to develop general methods to obtain well-defined protein and peptide assemblies, and to study multivalent interactions of these assemblies in a controlled fashion. In Chapter 2, a general synthetic strategy is described to obtain multivalent peptides and proteins using native chemical ligation. Different generations of poly(propylene imine) dendrimers were functionalized with N-terminal cysteine residues to allow the native chemical ligation reaction with C-terminal thioesters. Ligation of a peptide thioester with cysteine-functionalized dendrimers yielded multivalent peptide dendrimers of different generations with 4 to 16 peptides per dendrimer. This native chemical ligation strategy was expanded to recombinant proteins by employing intein-mediated protein expression and purification to obtain fluorescent proteins modified with a C-terminal thioester. Native chemical ligation of GFP-MESNA with a cysteine-modified dendrimer followed by ligation with peptide thioesters gives access to novel hybrid peptide-protein dendrimers. Ligation of 4 equivalents of GFP-MESNA with the cysteine-modified dendrimer yielded a branched, multivalent protein tetramer. Size exclusion chromatography combined with mass spectrometry proved to be an invaluable tool to study these complex bio-macromolecules. The use of surface plasmon resonance (SPR) biosensors enables real-time detection and monitoring of biomolecular binding events. Chapter 3 describes a chemoselective immobilization strategy for Biacore SPR sensor chips, based on native chemical ligation. First, a thioproline was introduced on the surface, which could be deprotected using mild conditions to an N-terminal cysteine residue. A streptavidin-binding peptide was immobilized via its C-terminus onto the biosensor chip, and subsequent binding experiments with streptavidin showed specific and reproducible binding to the peptide surface. Short ligation steps of peptide thioester were alternated with streptavidin binding experiments on a single chip. This provided an increased peptide loading after each ligation step, yielding enhanced protein-binding capacity. As an example of a recombinant protein, green fluorescent protein (GFP) was immobilized on the biosensor surface. Again, binding experiments with an antibody directed against GFP showed the specificity and robustness of the coupling strategy. The immobilization of S-peptide via native chemical ligation was used to illustrate the possibility of obtaining kinetic information from the specific interaction between S-peptide and S-protein. The presented approach allows for efficient immobilization of both recombinant proteins and synthetic peptides with high control over the degree of functionalization of the surface. In Chapter 4, native chemical ligation was used to synthesize multivalent peptides based on a streptavidin-binding peptide sequence derived from phage display. The synthetic multivalent scaffolds were used to mimic the multivalent character of the peptides on the head of a phage, without the presence of the phagemid coat proteins or genetic information. Peptides with different valency (from 1–4 copies per scaffold) and spacing were prepared and their affinity for streptavidin was measured using SPR. All multivalent peptides showed a significant increase in affinity compared to their monovalent counterpart and a binding model was used to describe the multivalent effect in a quantitative manner. However, the peptide dendrimers still showed considerably lower affinity than the streptavidin-binding phage. Possible reasons for this difference are discussed in this Chapter, as well as suggestions for further improvement of this dendrimer display by optimization of both scaffold rigidity and spacing of ligands. Although the covalent conjugation strategy described in Chapter 2 allowed the synthesis of tetravalent protein dendrimers of 110 kDa, non-covalent synthetic strategies are required for the development of even more complex protein assemblies. Chapter 5 explores the suitability of using the S-peptide–S-protein interaction to obtain well-defined, stable protein dendrimers. Association of S-peptide and S-protein results in the formation of an active enzyme, ribonuclease S, whereas neither fragment alone displays any enzyme activity. Native chemical ligation was used to couple four S-peptides via their C-terminal thioester to a cysteine-functionalized dendritic scaffold to yield a tetravalent S-peptide dendrimer. A fully functional ribonuclease S tetramer was prepared by addition of four equivalents of S-protein. Different biophysical techniques (ITC, SPR and mass spectrometry), and a fluorescent enzyme activity assay were used to quantify complex formation. For the non-covalent synthesis of more complex dendritic architectures, S-protein building blocks are required. Thioester-modified RNase A was obtained via recombinant expression as a precursor in the synthesis of multivalent S-protein assemblies. This noncovalent synthetic strategy based on ribonuclease S can be used to synthesize semisynthetic protein assemblies such as supramolecular polymers, gels and polymer networks with high control of structural organization, and may find applications in nanomedicine or functional biomaterials