Intermediate filament protein assembly : a proteomic approach
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Intermediate filament proteins (IFPs) form the main structural elements of a wool fibre. The IFPs of wool are comprised of two families; the acidic type I family and the neutral-basic type II family. During follicle development, one type I and one type II IFP develop into an obligate heteropolymer, which, through a series of associations with other heteropolymers, forms an intermediate filament. Two-dimensional polyacrylamide gel electrophoresis (20-PAGE) methods have been used to provide high-resolution separation of wool IFPs. Improvements in the method for maintaining reducing conditions and chaotrope constitution, combined with low % T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern, with numerous discrete minor spots not previously observed. Genomic studies have reported that there are eight genes which produce eight abundant IFPs in wool. It was hypothesised that the large number of additional spots seen on a 20-PAGE gel was due to post-translational modification (PTM) of the protein. Several common PTMs of proteins produce charge heterogeneity, including phosphorylation and glycosylation. However, analysis of wool IFPs by 20- PAGE techniques and mass spectrometry revealed no evidence of phosphorylation or glycosylation modifications. Conformational equilibria as a cause of protein charge heterogeneity has recently been reported. Investigations with both the type I and type II IFPs have shown that when single protein spots from a 20-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. The cause of this heterogeneity is thought to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension. This technique allowed the accurate assignment of IFPs resolved by 20-PAGE to protein families. Fractionation methods to separate the IFPs and intermediate filament associated proteins (IFAPs) were successfully developed. Further fractionation into the type I and type II IFPs was achieved along with partial success at isolating individual spots. In vitro assembly experiments with the different IFP families gives important information about the strength of different protein pairings. To date there are no reproducible, efficient, in vitro assembly conditions for keratinised wool IFPs. A comprehensive study to investigate assembly conditions for keratinised wool IFPs was undertaken. Assembly of filaments from IFPs was achieved after a partial digestion with chymotrypsin. Filaments were formed that varied in diameter from 10 to 40 nm, showing that higher ordered structures were being formed. This demonstrates that IFPs can be successfully assembled in vitro to form filamentous structures that may be able to be manipulated for biomaterial uses.