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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
34
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pubmed:dateCreated |
1998-9-14
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pubmed:abstractText |
Deletion mutants of human apolipoprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the function of the specific domains comprising residues 1-43, 1-65, 88-98, and 187-243, respectively, in the lipid-free conformation and in the lipid-binding mechanism of apo hA-I. Initial studies on apo Delta(1-43)A-I and apo Delta(187-243)A-I have already been reported. To aid purification of these mutants, a histidine-containing N-terminal extension was incorporated (+his); in cases where comparison with the (-his) construct was possible, little effect on the physical properties due to the (+his) extension was found. All mutants have folded structures in their lipid-free state, however these structures differ widely in their relative thermodynamic stability and extent of secondary structure. The mutant with the fewest residues deleted, apo Delta(88-98)A-I(+his), has the least secondary structure (only 34% helix) and is also the least stable (DeltaG = 2.9 kcal/mol). Determined from sedimentation velocity measurements on the lipid-free proteins, all but apo Delta(1-65)A-I(+his) exhibited a range of conformers in solution, which fluctuated around a highly elongated species (dimensions equal to approximately (14-16) x approximately 2.3 nm). Apo Delta(1-65)A-I(+his) exhibited a discrete species which was less asymmetric (dimensions equal to 9 x 2.9 nm). Apo Delta(88-98)A-I(+his) showed extreme heterogeneity with no predominating conformer. Spectroscopic studies (ANS binding and circular dichroism) indicate that there is little difference in the lipid-free structure of the carboxy-terminal deletion mutant, apo Delta(187-243)A-I(+/-his) compared to wild-type (wt) apo wtA-I(+/-his), but substantial differences are observed between wt and the amino-terminal deletion mutants, apo Delta(1-43)A-I, apo Delta(1-65)A-I(+his), and apo Delta(88-98)A-I(+his). In contrast, the lipid-binding properties are impaired for apo Delta(187-243)A-I(+/-his), as measured by dimyristoyl phosphatidylcholine (DMPC) liposome turbidity clearance kinetics and palmitoyloleoyl phosphatidylcholine (POPC) equilibrium binding. Apo Delta(1-43)A-I, apo Delta(1-65)A-I(+his), and apo Delta(88-98)A-I(+his) show lipid affinities statistically similar to apo wtA-I(+his), but significantly defective DMPC clearance kinetics. Interestingly, lecithin:cholesterol acyltransferase (LCAT) activation results correlate qualitatively with the lipid-binding affinity for all mutants but apo Delta(88-98)A-I(+his), suggesting that this mutant has an altered and possibly noncooperative lipid-bound structure as well as an altered lipid-free structure. These results suggest helix 1 (residues 44-65) and helix 10 (residues 220-240) are both required for native lipid-binding properties, while the presence of internal residues, at least helix 3 (residues 88-98), is essential for proper folding of both the lipid-free and lipid-bound conformations. Importantly, studies on apo Delta(88-98)A-I(+his) provide the first experimental evidence that a native-like structure is not necessary for native-like lipid affinity, but apparently is necessary for both DMPC solubilization and LCAT activation. These results provide support for a hypothetical, multistep structure-based mechanism for apo hA-I lipid binding.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Apolipoprotein A-I,
http://linkedlifedata.com/resource/pubmed/chemical/Factor Xa,
http://linkedlifedata.com/resource/pubmed/chemical/Histidine,
http://linkedlifedata.com/resource/pubmed/chemical/Lipids,
http://linkedlifedata.com/resource/pubmed/chemical/Peptide Fragments,
http://linkedlifedata.com/resource/pubmed/chemical/Phosphatidylcholine-Sterol...
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pubmed:status |
MEDLINE
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pubmed:month |
Aug
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pubmed:issn |
0006-2960
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
25
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pubmed:volume |
37
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
11714-25
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:9718294-Apolipoprotein A-I,
pubmed-meshheading:9718294-Circular Dichroism,
pubmed-meshheading:9718294-Enzyme Activation,
pubmed-meshheading:9718294-Factor Xa,
pubmed-meshheading:9718294-Histidine,
pubmed-meshheading:9718294-Humans,
pubmed-meshheading:9718294-Hydrolysis,
pubmed-meshheading:9718294-Lipid Metabolism,
pubmed-meshheading:9718294-Lipids,
pubmed-meshheading:9718294-Mutagenesis, Site-Directed,
pubmed-meshheading:9718294-Peptide Fragments,
pubmed-meshheading:9718294-Phosphatidylcholine-Sterol O-Acyltransferase,
pubmed-meshheading:9718294-Protein Binding,
pubmed-meshheading:9718294-Protein Folding,
pubmed-meshheading:9718294-Sequence Deletion,
pubmed-meshheading:9718294-Ultracentrifugation
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pubmed:year |
1998
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pubmed:articleTitle |
The lipid-free structure of apolipoprotein A-I: effects of amino-terminal deletions.
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pubmed:affiliation |
Department of Biochemistry and Molecular Genetics, Center for Macromolecular Crystallography, University of Alabama at Birmingham Medical Center 35294, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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