Reconstitution of Membrane Proteins or Subunits for Structural and Functional Studies
Date Issued
2007
Date
2007
Author(s)
Lin, Su-Ching
DOI
en-US
Abstract
Membrane proteins play significant roles in biological systems and function as
receptors, ionic channels, trans-membrane transporters, signal transducers, ion pumps
and free energy transducers, etc. In this dissertation, we focus on two membranes
proteins, membrane-associated progesterone receptors (MAPRs) and particulate
methane monooxygenase (pMMO). Steroid hormones can mediate rapid non-genomic
effects by binding to membrane-associated receptors. We have utilized the strategy of
sticky-end PCR for efficient cloning of the human liver MAPR in E. coli. BL21 (DE3)
strain, successfully purified and obtained two recombinant proteins, dubbed
pET21mapr and pGEX_mapr by using metal-affinity or/and glutathione S-transferase
affinity column. From spectroscopic studies and size-exclusion chromatography
analysis, the following results were obtained. The recombinant protein pET21mapr
exhibits α-helical structure both in the presence or absence of detergents. There is
strong hydrophobic interaction between detergent n-dodecyl-β-maltoside and
membrane-bound protein. The interaction could be used to decrease the oligomeric
state of proteins and alleviate protein aggregation. In CD studies, we show that the
hydrophobic steroid hormone progesterone protect the secondary structure of
progesterone receptor in the absence of detergent against the organic solvent DMSO,
which is typically used to deliver progesterone to the receptor. From 2D TROSY-based
VIII
1H-15N HSQC spectra, it appears that progesterone binds to the pET21mapr receptor at
residues of arginine, glycine and others. More NMR experiments are required before
the extract residues could be determined.
A second membrane protein is the pMMO enzyme from Methylococcus
capsulatus which consists of three subunits named pmoA, pmoB and pmoC. Subunit
pmoB is the largest one, and possesses two large N-terminal and C-terminal
water-exposed domains, which serve as the location of the E-clusters to provide a
reservoir of reducing equivalents for re-reduction of the C-clusters following methane
hydroxylation. In this dissertation, these recombinant pmoB proteins have to
successfully been cloned, expressed and purified. From Scatchard plots of copper
binding, we have concluded that the C-terminus water-exposed pmoBCW domain can
accommodate more than ten Cu(I) ions with dissociation constant 330μM , and that
the Cu(I) binding is highly cooperative (αH=4.3). These results clarify why the copper
ions are so readily lost during the purification process. In the x-ray structural analysis,
the preparation was devoid of ~10 copper ions, and did not possess biochemical
activity. In short, the preparation lacked the full complement of copper ions to be
functional. Thus, we have contributed to the understanding of the significance and the
role of the reduced E-cluster in the turnover of the pMMO.
Subjects
膜接合孕酮受質蛋白
賀爾蒙
紫質鍵結蛋白
微粒體甲烷單氧酵素
MAPRs
hormone
heme-binding protein
pMMO
E-cluster and C-cluster
Type
thesis
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