Please use this identifier to cite or link to this item:
Type: Thesis
Title: Folding and conformational stability of porcine growth hormone.
Author: Bastiras, Stan
Issue Date: 1992
School/Discipline: Dept. of Biochemistry
Abstract: The large scale production and purification of recombinant-derived porcine growth hormone (pGH) and mutants thereof, bream growth hormone (brGH) and human growth hormone (hGH) was carried out. Each of the protein products were analysed by various physicochemical means, in order to verify their identity and homogeneity. The equilibrium denaturation of pGH using the chemical denaturant guanidine hydrochloride (GuHCl), was monitored by a variety of spectroscopic and hydrodynamic probes. The denaturation of pGH resulted in absorbance- and fluorescence-detected transitions which were coincident, whereas far-UV circular dichroism- and hydrodynamic radius-detected transitions occured at higher concentrations of GuHCl, indicative of a greater stability to denaturation. Conformations intermediate to the folded and fully unfolded states were found to be stable at equilibrium under partially denaturing conditions. These intermediate forms have the characteristics of molten-globule or compact denatured states; a compact structure with considerable helix content, yet possessing a tertiary structure similar to that of the fully unfolded state. At concentrations above 10uM, the intermediate was shown to aggregate, forming a stable associated intermediate. These results suggest that pGH does not follow a simple two-state folding mechanism, but is consistent with the framework model of protein folding. The conformational stabilities of pGH, ten site-directed mutants of pGH, and wild-type bream and human GH, were determined using GuHCl-induced equilibrium denaturation under a standard set of conditions. Single amino acid changes in the sequence of pGH were shown to have different effects on (i) the conformational stability, (ii) the cooperativity of the denaturation transition, i.e., mGuHCl and (iii) the midpoint of the denaturation transition, i.e., [GuHCl] 1/2 . Bream GH was shown to have a stability similar to that of wild-type pGH whereas human GH, in accordance with previously published values [Brems, D. N., Brown, P. L., and Becker, G.W. (1990) J. Biol. Chem. 265, 5504-5511], was found to be significantly more stable than pGH and brGH. One mutant in which a methionine residue was replaced by a tryptophan [pGH(M8)], was found to be significantly more stable than wild-type pGH, due to an increase in both mGuHCl and [GuHCl] 1/2 coincidence of the UV-, fluorescence and hydrodynamic radius-detected equilibrium denaturation curves and the absence of significant amounts of associated forms suggests that pGH(M8) folding/unfolding is more closely approximated by a two-state mechanism than wild-type pGH.
Advisor: Wallace, John C.
Wells, Julian R.E.
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1992
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exception. If you are the author of this thesis and do not wish it to be made publicly available or If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
Appears in Collections:Research Theses

Files in This Item:
File Description SizeFormat 
01front.pdf354.14 kBAdobe PDFView/Open
02whole.pdf9.51 MBAdobe PDFView/Open

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