Accessing Specific Peptide Recognition by Combinatorial Chemistry

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

Ming Li

Molecular recognition is at the basis of all processes for life, and plays a central role in many biological processes, such as protein folding, the structural organization of cells and organelles, signal transduction, and the immune response. Hence, my PhD project is entitled “Accessing Specific Peptide Recognition by Combinatorial Chemistry”. Molecular recognition is a specific interaction between two or more molecules through noncovalent bonding, such as hydrogen bonding, metal coordination, van der Waals forces, π−π, hydrophobic, or electrostatic interactions. The association involves kinetic and thermodynamic states. (chapter 1) Molecular recognition is a complex and multifaceted process, which depends not only on the structure and functional groups of the molecules involved, but also on the environment. (chapter 1) Particularly in water, parameters such as pH, salt concentration and temperature, have a significant effect. (chapter1 & chapter 5) Affinity and selectivity are the two crucial aspects of molecular recognition. Generally, hydrophobic effects which reflect the change in water entropy on the hydrophobic surface upon complexation can increase affinity a lot; directional hydrogen bonding and van der Waals forces can enhance the selectivity between recognition pairs. (chapter 1) The degree of preorganization of the molecules can increase both the affinity and the selectivity of molecular recognition. (chapter 1, chapter 6 & chapter 8) These premises formed the basis of the present investigation. Combinatorial chemistry was invented in 1980s based on observation of functional aspects of the adaptive immune system. It was employed for drug development and optimization in conjunction with high-throughput synthesis and screening. (chapter 2) Combinatorial chemistry is able to rapidly produce many thousands fold more different compounds than conventional organic synthesis, which accelerate the process of discovering new drugs. (chapter 5, chapter 6 & chapter 8) It is a powerful approach in the process of obtaining recognition pairs with high affinity and selectivity. In order to study polar interactions in water, a very hydrophilic ligand library is built and its specific interactions with a very polar fully water soluble target, QTRTNTHRDG, was investigated in water. One ligand, EDYEVEEG, showed extremely specific and strong binding with this target. This peptide was obtained during bead-bead adhesion screening from the 78,125 membered library. (chapter 5) In addition, to investigate the influence of structural preference in recognition, a library with preorganized β-hairpin structures was built and its molecular recognition with a hexapeptide library was assessed by a “library-library” binding assay. Totally, 5.8×1011 possible molecular interactions could be assessed from a single screen. Several hits were isolated and the sequences obtained by MS-MS. Some of pairs showed nM range binding, which is equivalent to many protein-protein interactions. (chapter 6) Finally, to study molecular entanglement in recognition, a hook peptide library was built as sequence blocks containing two L-prolines, facilitating peptide back-bones to organize into a bend “hook” shape. Selection of pairs recognizing each other by entanglement with the conformational shape as a fundamental mechanism of molecular recognition was studied with this hook peptide library via the beadbead adhesion screening approach. The recognition pairs interlocked and formed a complex. (chapter 8) During accessing peptide molecular recognition by combinatorial chemistry, we faced several problems, which were solved by a range of analytical techniques including novel techniques and analysis devices. Bead-bead adhesion screening approach was utilized in all library screening in the present projects (chapter 5, chapter6 & chapter 8), and the results highlight the great advantage of this approach over other popular screening methods, e.g. those in which a solidphase receptor library is incubated with a ligand in solution. For MSMS identification the peptide from a single bead was released by 5% TEA aqueous solution. For hydrophilic sequences and MS analysis, in particular, aqueous TEA solution is superior to the conventional NaOH cleavage in providing sodium-free spectra for MS-MS. (chapter 5) Normally, when analyzing peptides by MS-MS, the peptide sequence should be less 15 amino acids long. To analyze a 21 membered hairpin peptide, MALDI and ESI approach were combined. (chapter 6) To remove false positive or medium strong hits, mechanical separation of is developed and used to select the best hits from the “hook” peptide library. The association strength of complex was also evaluated by MS-MS analysis. (chapter 8) a microchannel flow device was designed and utilized to measure binding constants on a single bead. (chapter 5) An important observation was that specific molecular interaction does not always provide NOEs resulting from short proton– proton distances and even with strong affinity to water molecules may participate in the complex formation. (chapter 5) The peptide recognition pairs with high affinity and specificity are extremely important for life science. β-hairpin tags are proteinogenic amino acid tags, which enablesv the introduction of the individual peptides into proteins by simple genetic fusion followed by a recombinant expression. (chapter 7) Hence, β-hairpin tags were used to purify over-expressed EGFP from lysed cell.
OriginalsprogEngelsk
ForlagDepartment of Chemistry, Faculty of Science, University of Copenhagen
StatusUdgivet - 2016

ID: 172389303