Recombinant human macrophage colony stimulating factor beta
(rhm-CSFβ) is a glycoprotein that stimulates the proliferation, differentiation
and survival of cells belonging to the monocyte-macrophage lineage. It
contains nine inter-subunit and intra-subunit disulfide bonds and represents
an excellent model system for studying disulfide bond formation during
protein folding because the assembly of its monomeric subunits and the
maturation of its biological activity depend on the progressive formation of
the correct disulfide structure during in vitro folding. Knowledge obtained
from these studies can be potentially useful in understanding the roles of
disulfide bond formation during protein folding in general.
rhm-CSF8 was modified by partial reduction of disulfide bonds,
yielding CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ. The modification did not affect the
biological activity, stability, or the overall conformation of the protein.
However, the C-terminal regions near the modification sites were shown to
exhibit faster deuterium exchange behavior as a result of the chemical
modification, indicating that the C-terminal regions became more flexible.
Folding kinetics of rhm-CSFβ and CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ were shown
to be essentially the same, suggesting that the modification did not affect
the folding kinetics of the oxidized rhm-CSFβ.
The denatured and reduced rhm-CSFβ was refolded with the aid of a
chemical oxidant. The data indicated that the in vitro folding rhm-CSFβ
proceeded via multiple pathways involving monomeric and dimeric
intermediates. Disulfide bond shuffling catalyzed by GSH/GSSG
represented an important isomerization step in folding. A dimeric
intermediate, D-SS8-cam2, was isolated and identified as a kinetic trap,
perhaps requiring significant structural arrangement to convert to the native
protein. The heterogeneous folding mixture detected by both disulfide bond
quenching and H/D pulsed labeling indicate that rhm -CSFβ folding is a
diffusion like process as described by the folding funnel model. / Graduation date: 2003
Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/37223 |
Date | 14 May 2002 |
Creators | Zhang, Yuan Heidi |
Contributors | Deinzer, Max L. |
Source Sets | Oregon State University |
Language | en_US |
Detected Language | English |
Type | Thesis/Dissertation |
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