Combined N- and C-Terminal Truncation of Human Apolipoprotein A-I Yields a Folded, Functional Central Domain
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- 作者：Jennifer A. Beckstead ; Brian L. Block ; John K. Bielicki ; Cyril M. Kay ; Michael N. Oda ; and Robert O. Ryan
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 22, 2005
- 年：2005
- 卷：44
- 期：11
- 页码：4591 - 4599
- 全文大小：152K
- 年卷期：v.44,no.11(March 22, 2005)
- ISSN：1520-4995
文摘
A combined N- and C-terminal truncation variant of human apolipoprotein A-I (apoA-I) wasdesigned, expressed in Escherichia coli, isolated, and characterized. Hydrodynamic experiments yieldeda weight average molecular weight of 34000, indicating apoA-I-(44-186) exists in solution predominantlyas a dimer. An axial ratio of 4.2 was calculated for the dimer based on sedimentation velocity experiments.Far-UV circular dichroism spectroscopy of apoA-I-(44-186) in buffer indicated the presence of 65%
-helix secondary structure. Guanidine hydrochloride denaturation experiments yielded a transition midpointof 0.5 M for apoA-I-(44-186). ApoA-I-(44-186) induced solubilization of dimyristoylphosphatidylcholinevesicles at a rate comparable to that of full-length apoA-I, displayed lipoprotein binding ability, and wasan acceptor of ABCA1-mediated cholesterol efflux from cultured macrophages. Fluorescence quenchingstudies with KI indicate that the three Trp residues in apoA-I-(44-186) are shielded from the aqueousenvironment. Taken together, the data indicate that lipid-free apoA-I-(44-186) adopts a folded conformationin solution that possesses lipid binding capability. The central region of apoA-I appears to adopt a globularamphipathic -helix bundle organization that is stabilized by intramolecular and/or intermolecular helix-helix interactions. Lipid association likely results in a conformational adaptation wherein helix-helixcontacts are substituted for helix-lipid interactions.
-helix secondary structure. Guanidine hydrochloride denaturation experiments yielded a transition midpointof 0.5 M for apoA-I-(44-186). ApoA-I-(44-186) induced solubilization of dimyristoylphosphatidylcholinevesicles at a rate comparable to that of full-length apoA-I, displayed lipoprotein binding ability, and wasan acceptor of ABCA1-mediated cholesterol efflux from cultured macrophages. Fluorescence quenchingstudies with KI indicate that the three Trp residues in apoA-I-(44-186) are shielded from the aqueousenvironment. Taken together, the data indicate that lipid-free apoA-I-(44-186) adopts a folded conformationin solution that possesses lipid binding capability. The central region of apoA-I appears to adopt a globularamphipathic -helix bundle organization that is stabilized by intramolecular and/or intermolecular helix-helix interactions. Lipid association likely results in a conformational adaptation wherein helix-helixcontacts are substituted for helix-lipid interactions.