Investigation into the structure and function of Hsp47

Collagen is the most abundant protein in mammals and is the main protein of connective tissue in animals [1]. It is a fibrous structural protein that is the major component of the extracellular matrix, which provides great tensile strength to ligaments, tendons, bone and skin, for example. Hsp47 is a procollagen and collagen specific chaperone that is thought to be involved in the correct assembly and transport of collagen chains. It is also a non-inhibitory member of the serine protease inhibitor superfamily and uses the pH-dependent mechanism, common to all members of this protein family, for binding and release to/from its substrate. A reduction in the pH of the environment triggers a conformational change in the serpin, which is vital for the function of Hsp47. It is this structural transition upon a change in pH that was exploited in the investigation into the regions of importance of for Hsp47-collagen binding. Most of the previous works examining the expression and function of Hsp47 have used mammalian forms of the protein and expression levels have proved problematic. This research studied Hsp47 in the poikilothermic vertebrate; Xenopus laevis, the ectothermic vertebrate; Danio rerio and the endothermic vertebrate; Mus musculus. Various constructs were tried to overcome the difficulty of low protein expression levels. Though there is high sequence homology between these species, each was observed as comprising of different characteristics in terms of protein expression, stability and biological activity. The typical pKa of the imidazole group of histidine is 6.2, which is also the approximate pH at which the Hsp47 and collagen interaction is perturbed [2]. Histidine residues are therefore thought to play a vital role in the conformational transition of Hsp47. Histidine variants were synthesised, whilst keeping in mind the regions of importance in the conformational change of other serpins, to identify possible binding sites and/or areas involved in the instigation of the structural change in Hsp47. The mutations allowed the first identification of a more stable serpin state of the protein and even more interestingly, forms that comprised of enhanced biological activity as they reduced collagen fibril formation. One of the mutants lacked any collagen binding ability, which may be an indication of a possible collagen binding site on Hsp47. The final aspect addressed in this research was protein crystallography. Previous attempts at Hsp47 crystal formation have proved unsuccessful due to the low expression and stability of the protein. This study used the newly identified, more stable form of Hsp47 to try to overcome these setbacks and produce Hsp47 crystals. The results presented in this thesis provide an insight into the function and structure of the non-inhibitory serpin Hsp47 and may enhance the understanding of the molecular mechanism by which the collagen-specific chaperone operates.