Queen's University - Utility Bar

QSpace at Queen's University >
Graduate Theses, Dissertations and Projects >
Queen's Graduate Theses and Dissertations >

Please use this identifier to cite or link to this item: http://hdl.handle.net/1974/5416

Title: Antifreeze Proteins: Activity Comparisons and De Novo Design of an Ice-Binding Protein
Authors: Yu, Sally Oi Wah

Files in This Item:

File Description SizeFormat
Yu_Sally_OW_201001_MSc.pdf4.8 MBAdobe PDFView/Open
Keywords: Antifreeze protein
Thermal hysteresis
Ice recrystallization inhibition
Ice-binding protein
Issue Date: 2010
Series/Report no.: Canadian theses
Abstract: Antifreeze proteins (AFPs) help cold-adapted organisms survive below 0 ◦C by binding to and inhibiting the growth of ice crystals. In this way, AFPs depress the freezing point of aqueous fluids below the melting point of ice (thermal hysteresis; TH). They also have the ability to inhibit ice recrystallization in the frozen state (ice recrystallization inhibition; IRI). Some AFPs show an order of magnitude higher TH activity than others, and are termed ‘hyperactive’. One of the objectives of this thesis was to see if IRI activities of the hyperactive AFPs are also an order of magnitude higher than the moderately active AFPs. Using a capillary-based assay for IRI, the activities of three hyperactive and three moderately active AFPs were determined. There was no apparent correlation between hyperactivity in TH and high IRI activity. However, mutations of residues on the ice-binding face (IBF) of both types of AFP reduced IRI and TH activities to a similar extent. In this way, the use of IBF mutant AFPs showed that the IBF responsible for an AFP’s TH activity is also responsible for its IRI activity. Analysis of the diverse AFP structures solved to date indicate that their IBFs are relatively flat, occupy a significant proportion of the protein’s surface area and are more hydrophobic than other surfaces of the protein. The IBFs also often have repeating sequence motifs and tend to be rich in alanine and/or, threonine. The de novo design of an ice-binding protein was undertaken using these features to verify the underlying physicochemical requirements necessary for a protein’s interaction with ice. Using site-directed mutagenesis, a total of sixteen threonine substitutions were made on one of the four faces of a cyanobacterial protein with no endogenous TH activity. The inclusion of eight paired threonines on one face of this quadrilateral helix gave the engineered protein low levels of TH activity, but at the cost of destabilizing the structure to some extent. The results of this study have validated some of the properties needed for the ice-binding activity of AFPs.
Description: Thesis (Master, Biochemistry) -- Queen's University, 2010-01-29 17:37:24.322
URI: http://hdl.handle.net/1974/5416
Appears in Collections:Queen's Graduate Theses and Dissertations
Biochemistry Graduate Theses (July 2007 - Sept 2016)

Items in QSpace are protected by copyright, with all rights reserved, unless otherwise indicated.


  DSpace Software Copyright © 2002-2008  The DSpace Foundation - TOP