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Strukturuntersuchungen verschiedener Mutanten des Maltose-Bindungsproteins (MBP) mit Hilfe der ESR Spektroskopie

  • Maltose binding protein (MBP) is a monomeric, two domain protein containing 370 amino acids. Seven double cysteine mutants of maltose binding protein (MBP) were generated with one each in the active cleft at position 298 and the second cysteine distributed over both domains of the protein. These cysteines were spin labeled and distances between the labels in biradical pairs determined by pulsed double electron–electron resonance (DEER) measurements. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structure of MBP without maltose and were found to be in excellent agreement. MBP is in a molten globule state at pH 3.3 and is known to still bind its substrate maltose. The ligand-binding affinity of the molten globule and the native states of MBP was studied by isothermal titration calorimetry. Ligand binding affinity measured by isothermal titration calorimetry for the native state of MBP was found to be comparable to that from the literature. Simultaneous measurements to investigate the molten globule state of MBP were implemented, including the use of far-and near-UV CD and the 8-anilino-1-naphthalene sulfonate (ANS) binding employing fluorescence techniques. Guanidine hydrochloride, urea and thermal denaturation studies have been carried out to compare the stability of the two states of maltose binding protein. In cw- experiments, the X-band EPR measurements at low temperature confirm indirect that all distances of the biradicals are above 20 Å, otherwise no evidence of dipolar interactions in the immobilized spectra were observed. DEER measurements of MBP in a molten globule state were yielding a broad distance distribution as was to be expected if there is no explicit tertiary structure and the individual helices pointing into all possible directions.

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Metadaten
Author:Mohammed Chakour
URN (permanent link):urn:nbn:de:hbz:386-kluedo-41602
Advisor:Wolfgang Trommer
Document Type:Doctoral Thesis
Language of publication:German
Publication Date:2015/08/21
Year of Publication:2015
Publishing Institute:Technische Universität Kaiserslautern
Granting Institute:Technische Universität Kaiserslautern
Acceptance Date of the Thesis:2015/06/18
Date of the Publication (Server):2015/08/21
Number of page:VI, 121
Faculties / Organisational entities:Fachbereich Chemie
DDC-Cassification:5 Naturwissenschaften und Mathematik / 540 Chemie
Licence (German):Standard gemäß KLUEDO-Leitlinien vom 30.07.2015