Self assembly of collagen mimetic peptides. (2017)
Type of ContentElectronic Thesis or Dissertation
Degree NameMaster of Science
PublisherUniversity of Canterbury
AuthorsMahapatra, Deeptishow all
This project is part of a wider research program that contributes to and focuses on the utilization of lower value red meat components to create new meat-derived foods and ingredients. There have been significant advances in studying primary, secondary, tertiary and quaternary structures of food proteins in recent years but still, there are fundamental gaps in understanding the interactions of proteins and peptides in whole and reconstituted foods.
Collagen has a profound impact on food texture and this thesis focuses on analyzing collagen at the molecular level using collagen model peptides, to better understand self-assembly in meat derived food products. Short peptides are easy to design and synthesize, making them an excellent model system for studying self-assembly. Extensive literature survey has been done to understand self-assembly with short peptides, in Chapter 1. The materials and the methodologies adopted in this thesis have been dealt with in Chapter 2.
In Chapter 3 the effect of varying chain lengths of GPO peptides on triple helical formations and triple helical stabilities have been studied. After the characterization of triple helical assemblies and supramolecular assemblies, the most significant findings were - (1) the triple helix formation was found to be directly proportional to chain length of the peptide, concentration of mixture and time of incubation (2) longer chain lengths of peptides exhibited higher triple helical stabilities (3) triple helical formations and stabilities were supported in high salt environments (4) pH 7 favoured triple helical stability the most followed by pH 3.5 (5) self-association was observed to be limited by the low concentration of monomers.
The effect of temperature cycles on self-assembly of collagen peptides has been studied in Chapter 4. Infrared (IR) spectroscopy was used to study the changes brought about in the underlying polyproline II chains and in intra and inter hydrogen bonding patterns of the supramolecular assemblies and crystals of the GPO peptides. From techniques like AFM & DLS, heat treatment was seen to cause reduction in size of the GPO peptide aggregations. It is speculated that the shrinkage of these supramolecular assemblies were a result of breakage of the intra and inter helical H bonds after heat treatment. D-spacing in (GPO)10 and (GPO)7 peptides were found to be exactly the same, 12.26 Å, which indicates that there were similarities in packing for both triple helices. The D-spacing in the (GOO)10 peptides was ~ 12.36 Å that suggests a different staggered pattern for the helical arrangement and hence a different supramolecular assembly pattern.
Examination of triple helical packing of GPO peptides with crystallography has been conducted in Chapter 5. Collagen model peptides of varying GPO chain lengths have been successfully crystallized. Racemic (GPO)5-Gly peptides have been studied. The X-ray crystal structure of the (GPO)10 peptide is solved at a resolution of 0.89 Å. Apart from generic features seen in the previously solved structures, detailed findings from this structure were - (1) proline rings can adopt two stable conformational states (2) the helical pitch was seen to vary around a 7/2 helix and (3) the stabilization of the crystalline network was water-mediated via hydroxyproline hydration, which bridges hydrogen bonding of helices.
The effect of oxidation on triple helices and supramolecular assemblies has been studied in chapter 6. (GOO)10 peptides were characterized to understand the differences occurring in the assembly properties of GPO and GOO collagen peptides. This was done to help explore possibilities of improving the food matrices. (GOO)10 peptides were found to be more stable than the (GPO)10 peptides. In second part of the chapter, the impact of heating (cooking) on self assembly of (GPO)7 collagen model peptides has been investigated. Tandem mass spectrometric analysis has been carried out to sequence these peptides and characterize the oxidative modifications occurring in amino acid sites in the peptide chain. Middle parts of the (GPO)7 peptide sequence have been found to be highly prone to oxidation.
Apart from bridging gaps in studies of oxidative modifications in red meat processing, in the food industry, this research on self assembly of collagen model peptides will also prove beneficial in medical applications like drug delivery, tissue engineering, biomedical applications related to collagen etc.