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Understanding the prion protein

Understanding the prion protein

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This report will explore two aspects which may influence the Prion Protein behaviour- protein stability and phosphorylation. Inroduction of additional favourable
interactions at positions 129 and 199 of the protein will stabilize the overall structure of the PrP. Presence of phosphorylation sites on the surface of the PrP may indicate that kinase enymes are potential interacting partners of the
protein.
This report will explore two aspects which may influence the Prion Protein behaviour- protein stability and phosphorylation. Inroduction of additional favourable
interactions at positions 129 and 199 of the protein will stabilize the overall structure of the PrP. Presence of phosphorylation sites on the surface of the PrP may indicate that kinase enymes are potential interacting partners of the
protein.

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Published by: Undergraduate Awards on Sep 01, 2012
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11/14/2013

 
Understanding the Prion ProteinTatiana Papkovskaia05354071Understanding the mechanisms of thePrion Protein (PrP) associated processes isessential for understanding its function. Thisreport will explore two aspects which mayinfluence the PrP behaviour-protein stability andphosphorylation. These topics were chosen on thebasis of the following hypotheses.Hypothesis 1: Inroduction of additional favourableinteractions due to the presence of alternativeresidues in positions 129 and 199 of the proteinwill stabilize the overall structure of the PrP.Hypothesis 2: Presence of phosphorylation siteson the surface of the PrP may indicate that kinaseenymes are potential interacting partners of theprotein.IntroductionMisfolded forms of the Prion Protein (PrP)are believed to be the primary cause of a numberof neurodegenerative disorders. The mechanismfor the development of such diseases is not fullyunderstood but is thought to be linked to theconformational rearrangement within the proteinstructure. Both PrPc (native form) and PrPsc(infectious form) are produced by the same gene.Experiments have shown complete sequenceidentity and similar levels of mRNA for bothproteins (1). A key difference between the twoisoforms is the tertiary structure of the foldedmolecules. Cellular PrP contains an extensivehelical arrangement while the PrPsc structure isdominated by beta sheets. The ability of PrPsc toassociate via the beta sheet structures results in theformation of aggregates that can initiate andaccelerate the disease (1). Aggregation in the formof plaque formation is the main feature of Priondiseases which leads to the decline in cognitiveand motor functions eventually resulting in death.Protein folding is a highly specific andregulated process which assembles the primarypeptide structure into the physiologically relevantnative state. This process is believed to occurthrough a rapid trial and error until the mostthermodynamically stable configuration isBIOC40050achieved. The search funnels the variable
 
confirmations to a transition state believed to bethe point at which the critical residues within theprotein structure assemble together to form afolded nucleus (2). The rest of the proteinstructure can then rapidly form around thisnucleus. The rate of formation as well as the finalstructure of the protein is determined by thecorresponding amino acid sequence. The specificassociation of the individual residues results in theformation of the stable molecule. Mutationswithin the sequence can thus affect the fold andstability of the resulting structure (3).The onset of misfolding diseases isthought to be initiated by the increase in thepopulation of the partially unfolded states whichleads to the propensity of the protein to aggregate(4). In Prion associated diseases a conformationalchange is believed to be responsible for theformation of the misfolded PrP. (5). At themoment two proposed mechanisms for theconformational conversion exist (6). One proposesa nucleation dependent polymerisation where thePrPsc is less stable than PrPC. This process isbelieved to be dependent on the PrPscconcentration which when elevated forms aseeding nucleus promoting conversion andaggregation. Alternatively the other processsuggests PrPsc to be more stable than PrPc.Conversion takes place via a destabilizedintermediate where PrPsc binding (monomericassociation) helps to overcome the transition stateenergy barrier. The purpose of this study is todetermine how mutations in critical regions of theprotein affect the overall stability of the PrPc.Determination of structural stability ofproteins has proven to be a difficult task due to thedifferent energy terms that contribute to the freeenergy equation (7). The contribution of structuralalterations to energy changes have been used todesign algorithms which calculate the change inthe stability of proteins. The Uffbaps server is anexample of such a programme. It will be used inthis study to determine how mutations within thePrP structure affect the stability of the protein bycalculating the difference in the free energybetween the mutant and the WT. These
 
observations will then be supported with structuralinformation from Pymol and YASARA.The activity and localization of manyproteins is often controlled by regulatorymechanisms such as post translationalmodification. Protein phosphorylation preformedby the kinase enzymes can alter the behaviour andconfirmation of a specific substrate which can inturn lead to activation or inhibition of downstreamevents. This type of post translationalmodification can occur in a number of differentcellular compartments and usually results inswitching the activity of the protein from one stateto another (8). Sequence recognition by thekinases is believed to be a huge factor indetermining substrate specificity (9). A number ofanalytical methods have been developed in anattempt to predict possible interacting partners andusing this information to identify unknownfunctions. Sequence motifs involving 7-12 aminoacids are believed to surround the acceptor residueand contact the kinase active site prior tophosphorylation (10). Slight variation within thesesequence motifs allows some kinases to havenumerous interacting partners. NetPhosphoK is aprogramme which utilizes a computer algorithm tosearch for possible sites for kinase recognition.While accurate predictions can be made onwhether a protein contains phosphorylation motifsthese predictions are not always biologicallyrelevant. Intracellular localization, processing andsurface accessibility are just some of the factorswhich can determine whether a substrate willassociate with the appropriate enzyme and bemodified. Furthermore the predicted motifs canoccur at random in non modified proteins whichcan result in false positive predictions (11).The true biological function of the PrionProtein is still unclear. Protein function cansometimes be predicted through identification ofinteracting partners. To date no enzymes targetingthe PrP for phosphorylation have been identified.There are no phosphorylation site entries on thePhospho.ELM databasehttp://phospho.elm.eu.org/. This study willattempt to identify regions of the PrPc which maybe subject to phosphorylation using the PhosphoKprogramme. Five of the top scores will beexamined. The predicted residues will then bemapped on the protein structure using Pymol.Their accessibility and likelihood of occurrencewill then be assessed.Materials and methods:-The mutants were designed in YASARA byreplacing the residue of interest with the desired

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