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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Kristína Prokešová Supervisor: doc. PharmDr. Miroslav Miletín, PhD. Consultant: Dr. Richard Wombacher Title: Synthesis of "Chemical Tags" and their application for selective protein labelling This diploma thesis is aimed on the synthesis and application of chemical tags. In theoretical part, protein labelling in general is discussed and fluorescent proteins, as routinely used technique for protein tracking, are shortly presented. The greatest attention is dedicated to chemical tags which consist of genetically encoded protein or peptide tag fused to the protein of interest (POI) and from a small fluorescent molecule which labels the POI-tag fusion. Particular representatives with their advantageous and disadvantageous properties are mentioned and super-resolution microscopy and calcium imaging, as applications of chemical tags, are explained. Experimental part is divided into chemical synthesis and biological methods. In synthetic part, four precursors of chemical tags have been prepared - two precursors of TMP-tag small molecule and two precursors of a chemical calcium dye. These precursors can be connected to create a new TMP-tag applicable for calcium imaging. One...

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 161-195). Also available in electronic version.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Anne S. Robinson, Dept. of Chemical Engineering. Includes bibliographical references.

Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs

Going down the folding funnel, proteins may sample a wide variety of conformations, some being outright detrimental to the organism. Yet, the vast majority of polypeptide molecules avoid such pitfalls. Not only do they reach the native minimum of the energy landscape; they do so via blazingly fast, biased, routes. This specificity and speed is remarkable, as the surrounding solution is filled to the brim with other molecules that could potentially interact with the protein and in doing so stabilise non-native, potentially toxic, conformations. How such incidents are avoided while maintaining native structure and function is not understood.  This doctoral thesis argues that protein structure and function can be separated in the folding code of natural protein sequences by use of multiple partly uncoupled factors that act in a concerted fashion. More specifically, we demonstrate that: i) Evolutionarily conserved functional and regulatory elements can be excised from a present day protein, leaving behind an independently folded protein scaffold. This suggests that the dichotomy between functional and structural elements can be preserved during the course of protein evolution. ii) The ubiquitous charges on soluble protein surfaces are not required for protein folding in biologically relevant timescales, but are critical to intermolecular interaction. Monomer folding can be driven by hydrophobicity and hydrogen bonding alone, while functional and structural intermolecular interaction depends on the relative positions of charges that are not required for the native bias inherent to the folding mechanism. It is possible that such uncoupling reduces the probability of evolutionary clashes between fold and function. Without such a balancing mechanism, functional evolution might pull the carpet from under the feet of structural integrity, and vice versa. These findings have implications for both de novo protein design and the molecular mechanisms behind diseases caused by protein misfolding. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Proteins of sunflower seed