Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488090
Title: Theoretical and experimental studies of mucin peptides and glycopeptides
Author: Wakefield, Matthew Jerome
Awarding Body: University of Manchester : University of Manchester
Current Institution: University of Manchester
Date of Award: 2003
Availability of Full Text:
Access from EThOS:
Abstract:
Mucins are very large (tens of MDa) molecules that provide the primary structural components of mucus gels functioning in the Human airways, eyes, reproductive tracts and gut. The majority of the mucin macromolecule consists of repeated sequences of heavily O-glycosylated peptide that is rich in serine (Ser) andlor threonine (Thr), and proline (Pro). These regions of glycoprotein give gel-forming mucins characteristically extended conformations, often exceeding lOf.Ull length, that interweave to form the gel matrix. The nature of this extended conformation poses a conceptual problem as to how these molecules are handled through the biosynthetic pathway. The final stage of biosynthesis involves packaging of the mucins for storage within secretory granules in a highly condensed state. De-condensation of the mucin polymer network upon exocytosis occurs at such a rate «20ms to reach hydrated volume) as to suggest that the condensed phase must be highly organised. In order to begin to understand this condensation/de-condensation process, but also the structure/function relationship, of mucins it is important to understand the conformational ensemble available to these glycoproteins. Using a theoretical approach, supported by experimental evidence, this study aims to improve the understanding of the conformational dynamics of mucin peptides and glycopeptides. Short molecular dynamics (MD) simulations of sequences from MUC2 and MUC7 were used to begin to understand the preferred conformations for Thr, Ser and Pro residues and the related intramolecular hydrogen bond interactions in peptides. Long MD simulations, of 20ns in duration, were then performed on model peptide sequences to further investigate these observations. These simulations allowed us to build up an understanding of the role that water plays in determining the conformation of these biomolecules. Furthermore the effect of peptidyl-prolyl isomerisation on molecular conformation was investigated. In order to understand the effect of O-glycosylation on the conformational dynamics of the peptide, these simulations were contrasted to a series of simulations on the same model peptides with the addition of a-linked N-acetylgalactosamine (GaINAc). Preferred conformations of the alinkage region were investigated, along with the resulting sugar to peptide hydrogen bond networks. In order to investigate the effect of the a-linked GalNAc a series of simulations were performed with 'un-natural' ~-linked GalNAc addition to the model peptides. Hydrodynamic, viscosity and circular dichroism (CD) are techniques that aid the interpretation of the structural content of biomolecules, and were applied to help understand the conformational properties of a series of mucin synthetic peptides and poly amino acids. The MD results show that mucin peptides and model peptides rich in Ser, Thr and Pro have conformational ensembles dominated by an extended yet flexible peptide backbone, an observation supported by hydrodynamic and viscometry studies. The conformational equilibrium found in MD favours the Pn (poly-Pro II helix) secondary structure, a prediction that affords some support from CD studies. Experimental data for poly-Thr molecules ranging from tens to hundreds of residues in length display a compact structure, which reveals an unusual secondary structural content on examination by CD. These observations allow a model to be built of how mucin sequences may exist in compact and extended conformations, necessary for the condensation/de-condensation process. Analysis of MD simulations of model mucin glycopeptides, modified with a-linked glycan, reveal a highly dampened dynamic motion in terms of the amplitude and frequency of shifts in the molecular end-to-end distance vector. The a-linked GalNAc are clearly seen to exist in a set of stable conformers having specific networks of intramolecular hydrogen bonds that shield the peptide backbone from proteolytic attack. This shielding maintains the structural integrity and therefore the function of the mucin macromolecule. MD investigation of p-linked GalNAc reveals a chaotic conformational dynamic associated with the linkage region and a lack of stable intramolecular interactions. Such observations highlight the fact that a-links were selected over p-links by the O-glycosylation machinery in nature.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.488090  DOI: Not available
Share: