A mutational analysis of the hinge region of the N-lobe of lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

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dc.contributor.authorShewry, Steven Christopher
dc.date.accessioned2018-02-26T22:40:12Z
dc.date.available2018-02-26T22:40:12Z
dc.date.issued1996
dc.description.abstractLactoferrin is an 80kDa iron binding glycoprotein that is found as a major component of human milk, as well as in many other exocrine solutions. Lactoferrin binds reversibly, and with high affinity, 2 Fe3+ ions with 2 synergistic C032- ions. The crystal structure shows that the polypeptide chain is folded into two similar lobes, each binding one Fe3+ and CO32- ion. In the metal free state, the N-terminal lobe has been found to adopt an open structure, with rotation occurring around two residues found in separate beta strands located at the back of the binding site in a region referred to as the hinge. A sequence alignment of these two strands over the greater transferrin family shows a very high level of conservation particularly of the two residues at the centre of this rotation (Pro 251 and Thr 90). The N-terminal half of human lactoferrin (LfN) has been constructed, expressed and the crystal structure determined. In an attempt to understand the importance of the conservation of these two residues, and their effect on binding, a mutational analysis was initiated. Oligonucleotide site-directed mutagenesis has been used to construct mutants in the cDNA encoding for human lactoferrin using the M13 bacteriophage. The mutant cDNA was transformed into a mammalian expression vector (pNUT). After transfection of the pNUT vector into baby hamster kidney cells (BHK), the mutant proteins were expressed and purified from the culture medium using a CM-sephadex ion-exchange column. Absorption maxima and pH-dependent iron-release experiments were carried out on the mutants. The data shows that the mutants behave essentially the same as LfN, the exception being P251G which appears to release the iron over a shorter pH range. The reason for this is not yet fully understood. The crystal structure of P251A in the iron-bound form was solved by molecular replacement using the structure of LfN as the starting model. The structure of P251A was refined using data between 20.0 and 2.0 วบ. The current model has good geometry and has an R-factor of 18.6 %. Analysis of the structure shows that it is essentially identical to that of the LfN structure. Although the structure of the iron-free form has not been determined, it appears that changes to the hinge region of the N-lobe of lactoferrin do not affect the iron-binding or structural characteristics of the protein.en_US
dc.identifier.urihttp://hdl.handle.net/10179/12822
dc.language.isoenen_US
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectLactoferrinen_US
dc.titleA mutational analysis of the hinge region of the N-lobe of lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey Universityen_US
dc.typeThesisen_US
massey.contributor.authorShewry, Steven Christopher
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorMassey Universityen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M. Sc.)en_US
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