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Structural characterization of the leukemia drug target ABL kinase and unfolded polypeptides by novel solution NMR techniques

Vajpai, Navratna. Structural characterization of the leukemia drug target ABL kinase and unfolded polypeptides by novel solution NMR techniques. 2010, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9003

Abstract

Novel weak alignment techniques and new biochemical strategies like selective isotope labeling in combination with other high-resolution solution NMR methods have been applied to characterize folded and unfolded polypeptides. This includes the characterization of the solution conformations of the leukemia drug target Abelson (ABL) kinase in complex with three clinical drugs (imatinib, nilotinib and dasatinib), unstructured/urea-denatured polypeptides, and the transcriptional repressor in the highly conserved Notch pathway, HES1.
Solution NMR studies of ABL kinase in complex with three clinical inhibitors
Aberrant forms of ABL kinase are important drug targets for the treatment of chronic myelogenous leukemia (CML). The results of this thesis provide the first detailed characterization of solution conformations of ABL tyrosine kinase in complex with three effective clinical inhibitors imatinib, nilotinib and dasatinib. In solution, a centrally located regulatory segment termed the activation loop adopts the non-ATP binding inactive conformation in complex with imatinib and nilotinib, and preserves the ATP-binding active conformation in complex with dasatinib. However, relaxation studies and/or line broadening of some resonances in the activation loop and the phosphate-binding loop (P-loop) indicate presence of microsecond to millisecond dynamics for all the investigated ABL-inhibitor complexes. These results contribute to our understanding of drug resistance and support the rational design of improved kinase inhibitors (Manley et al., 2006, Vajpai et al., 2008a, Vajpai et al., 2008b).
Conformational studies of unstructured oligopeptides by residual dipolar couplings
The characterization of unfolded states of polypeptide chains is of high significance with regard to their role in biological processes and to understanding protein folding. Here, we have investigated the influence of amino acid substitutions X on the conformation of unfolded model peptides EGAAXAASS as monitored by backbone RDCs. The RDCs show a specific dependence on the substitutions X that correlates to steric or hydrophobic interactions with the adjacent amino acids. RDC profiles along the nonapeptide sequence show large variations for a few amino acid substitutions. In particular, RDCs for glycine and proline indicate less or more order than the other amino acids, respectively. The RDCs for aromatic substitutions tryptophan/tyrosine or isoleucine give evidence of kink or stiffness in the polypeptide backbone (Dames et al., 2006).
For a quantitative description, these experimental results were compared to the predictions from the statistical coil model, which derives amino acid specific local conformations from the torsion angle distribution of non-, non- structures in folded proteins, or all-atom molecular dynamics (MD) simulations. While the coil model reproduced, to some extent, the observed RDC pattern for most substitutions, MD simulations showed stronger deviations from the experimental data. This indicates specific deficiencies in both the statistical coil model and the MD simulations. For the coil model, the discrepancy may be related to imperfect modeling of the side chains, while for MD simulations, inadequate sampling of the conformational space in the time used for the simulations may be the most plausible reason.
Side-chains conformations in urea-denatured proteins: a study by 3J scalar couplings and residual dipolar couplings
In order to probe the conformational behavior of the side-chains in unfolded states, we have measured an extensive set of six three-bond scalar couplings (3JNH, 3JC’H and 3JHH) and two 1DCH residual dipolar couplings (RDCs) on urea-denatured proteins, ubiquitin and protein G. Interpretation of the 3J couplings by a model of mixed staggered χ1 rotamers yields excellent agreement and also provides stereoassignments for 1Hβ methylene protons. Independent analysis of 1DCH RDCs obtained in polyacrylamide gels show good correlation with the RDCs predicted from the 1 populations obtained from the 3J data and a coil model ensemble of 50000 conformers according to the coil library backbone angle distribution. The study validates coil model as a good first approximation of the unfolded state. However, individual variations from the coil averages of up to 40% are highly significant and must originate from sequence- and residue-specific interactions. The deviations between the measured and predicted values also indicate that the local backbone geometries may be improved by incorporation of the additional RDC information (Vajpai et al., 2010). Backbone resonance assignment of the 31 kDa of homodimer of apo-HES1
HES1 acts as an effector of highly conserved intercellular Notch signaling pathway by repressing the expression of target genes. The backbone resonance assignment and homology modeling of the 31 kDa homodimer of apo-HES1 are reported. The obtained results are being used for further structural studies on HES1.
Advisors:Grzesiek, Stephan
Committee Members:Jahnke, Wolfgang
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
Item Type:Thesis
Thesis no:9003
Bibsysno:Link to catalogue
Number of Pages:139 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:30 Apr 2010 10:53

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