Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2004. / Härtill 4 uppsatser.

C-terminal binding proteins (CtBPs) are transcriptional co-repressors involved in developmental processes, and also implicated in a number of breast, ovarian, colon cancers, and resistance against cancer chemotherapy. CtBP is a validated novel potential anti-cancer target. In this project we sought to develop potent and selective small-molecule inhibitors of CtBP. Using a combination of classical medicinal chemistry and modern computational approaches, we designed a potent inhibitor HIPP (hydroxyimino-3-phenylpropanoic acid) that showed an IC50 of 0.24 μM against recombinant CtBP. Further elucidation of the structure-activity relationship (SAR) of HIPP led to the design of more potent inhibitors 3-Cl HIPP (CtBP IC50 = 0.17 μM) and 4-Cl HIPP (CtBP IC50 = 0.18 μM). These compounds also showed inhibition in HCT-116 colon cancer cells with GI50 values ~ 1-4 mM. The compounds showed no off-target toxicity against a closely related protein. This is a starting point for the development of CtBP inhibitors as anti-cancer therapeutics. The second part of this dissertation focuses on C–H activation chemistry. C–H activation is the most atom-economical method of introducing complexity into a molecule, even at late stages of drug/product development. We have used solid-supported palladium nanoparticle catalyst (Pd-MWCNT) to investigate the scope of C–H activation reactions it can catalyse. Pd-MWCNT was found to efficiently catalyse N-chelation directed C-H activation reactions – halogenations, oxygenations and arylations. The turn-over numbers for these reactions were significantly higher than that of the reported homogenous catalyst. The added advantages of reuse/recyclability of catalyst, low contamination of metal in the final product make this catalyst very attractive on an industrial scale. This work serves as a foundation for the further development of Pd-MWCNT catalyst in late-stage synthesis of drugs and/or diversification of products.

No description available.

Zhang, Bin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 154-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques.</p><p>The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles.</p><p>Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins.</p><p>We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB).</p><p>The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism.</p><p>The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.</p>

<p>DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. </p><p>To expand the knowledge of protein–DNA recognition, we have utilized<i> in vitro</i> evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. </p><p>Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring <i>K</i>_D values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.</p><p>mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. </p><p>The two <i>in vitro</i> selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.</p>

DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. To expand the knowledge of protein–DNA recognition, we have utilized in vitro evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring KD values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure. mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. The two in vitro selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.

Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques. The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles. Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins. We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB). The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism. The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.

Most amino acid sequences are predicted to specify a single three-dimensional protein structure. However, the identification of "metamorphic" proteins, which can adopt two folds from a single amino acid sequence, has challenged the one sequence/one structure paradigm. Polar-to-hydrophobic substitutions have been suggested computationally as one mechanism to decrease structural specificity, allowing the population of novel folds. Here, we experimentally investigate the role of polar-to-hydrophobic substitutions on structural specificity in the homodimeric ribbon-helix-helix protein Arc repressor. Previous work showed that a single polar-to-hydrophobic surface substitution in the strand region of Arc repressor (Arc-N11L) populates the wild-type fold and a novel dimeric "switch" fold. In this work, we investigate an Arc repressor variant with the N11L substitution plus two additional polar-to-hydrophobic surface substitutions (Arc-S-VLV). We determine that this sequence folds into at least three structures: both dimer forms present in Arc-N11L, and a novel octamer structure containing higher stability and less helicity than the dimer folds. We are able to isolate and stabilize a core of the S-VLV octamer by limited trypsinolysis and deletion mutagenesis (Arc-VLV 4-44). The shortened construct contains only the octameric structure by removing disordered C-terminal segments nonessential for this fold. A two-dimensional NMR spectrum of VLV 4-44 and subsequent trypsinolysis of this construct suggests that at least two types of subunits comprise the S-VLV octamer: subunits structured from residues 4 to 44 and subunits structured from residues 4 to 31. Crystal trials of trypsinolyzed Arc-VLV 4-44 yielded several leads, suggesting that obtaining a high resolution structure of the S-VLV octamer is possible. Relatedly, we determine that the proline residues flanking the Arc repressor strand act in concert as "gatekeepers" to prevent aggregation in the S-VLV sequence. We also find that three highly hydrophobic surface substitutions in the Arc repressor strand region are necessary and sufficient to promote higher-order oligomer formation. In summation, this work reveals in an experimental context that progressive increases in polar-to-hydrophobic surface substitutions populate increasingly diverse, structurally degenerate folds. These results suggest that "metamorphic" as well as "polymetamorphic" proteins, which adopt numerous folds, are possible outcomes for a single protein sequence.