Probing the Lipid-Free Structure and Stability of Apolipoprotein A-I by Mutation

Gorshkova, Irina N.; Liadaki, Kalliopi; Gursky, Olga; Atkinson, David; Zannis, Vassilis I.

doi:10.1021/bi0014406

Cited by 49 publications

(93 citation statements)

References 47 publications

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“…(30). A mutation G185P/G186P located in the Cterminal part of apoA-I affects fluorescence of tryptophans located in the N-terminal half of apoA-I, which suggests a close proximity of the N-and C-termini of apoA-I and their interaction with each other in solution (29). Substitutions L222K/F225K/F229K (29) and E125K/E128K/K133E/E139K (30) stabilize lipid-free apoA-I and increase its α-helical content, while substitutions V156K/A158E (45) and E110A/E111A (12) destabilize the protein and result in a loss of helical structure.…”

Section: Discussionmentioning

confidence: 99%

“…Far-UV CD spectra were recorded as described previously (29,30). For each protein, spectra were recorded at several protein concentrations within the indicated range and then normalized to molar residue ellipticity.…”

Section: Circular Dichroism Spectroscopymentioning

confidence: 99%

“…However, even with this considerable amount of accumulated data, the structural details of the lipid-free and lipid-bound conformation are not fully defined and experimental results leading to definition of residues involved in stabilizing the protein lipid-free and lipid-bound conformations are not sufficient. So far, there have been no reported experimental data demonstrating that electrostatic interactions between specific basic and acidic residues located in two anti-parallel apoA-I molecules on the edge of rHDL are essential for the stabilization of the discs, as predicted by the double belt model (27).Studies of bioengineered mutant apoA-I forms remain efficient to obtain information that leads to detailed understanding of the conformation and structure-function relationships of apoA-I (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)24,29,30). Our earlier study (29) using mutation to probe the conformation and stability of the C-terminus of apoA-I suggested the presence of stable helical structure in the region between residues 165 and 243 and a close proximity of the N-and C-termini in the lipid-free protein.…”

mentioning

confidence: 99%

“…Studies of bioengineered mutant apoA-I forms remain efficient to obtain information that leads to detailed understanding of the conformation and structure-function relationships of apoA-I (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)24,29,30). Our earlier study (29) using mutation to probe the conformation and stability of the C-terminus of apoA-I suggested the presence of stable helical structure in the region between residues 165 and 243 and a close proximity of the N-and C-termini in the lipid-free protein.…”

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confidence: 99%

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Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

et al. 2006

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To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Δ(61-78) and Δ(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid:cholesterol:apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4, and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL, but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation suggesting involvement of R160/H162 in important inter-helical interactions. The results on the Δ(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by electrostatic inter-helical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoprotein (HDL) that plays a key role in the biogenesis and atheroprotective functions of HDL and in reverse cholesterol transport (1-3). Lipid-free apoA-I secreted by cells can interact functionally with ATP binding cassette transporter 1 (ABCA1) to promote efflux of cholesterol and phospholipids from cells. This process leads to the lipidation of apoA-I and formation of discoidal HDL (4,5). ApoA-I in discoidal HDL activates the enzyme lecithin:cholesterol acyltransferase (LCAT) converting cholesterol to cholesterol ester and thereby promoting the maturation of HDL particles from discoidal to spherical. ApoA-I bound to discoidal and spherical HDL also interacts functionally with scavenger receptor class B type I (SR-BI) (4, 6,7). On binding HDL, SR-BI mediates selective uptake of cholesterol esters and other lipids χ This work was supported by PO1HL 26335 and grant HL 48739 from the National Institute of Heath *To whom correspondence should be addressed at Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, W-322, Boston, MA 02118. Fax: (617) from HDL by cells and net efflux of excess cholesterol (4,6). In the course of apoA-I metabolism, the protein function is modulated by alterations in its structure and stability (5,(8)(9)(10)(11)(12)(13)(14)(15). Therefore, detail...

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Section: Discussionmentioning

confidence: 99%

Section: Circular Dichroism Spectroscopymentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

et al. 2006

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“…A continuous helix repeat assignment describes 10 tandemly repeated helices punctuated mainly by prolines. However, although this continuous helical model is the most widely used one, assignment of the precise secondary structural distribution throughout the apoA-I sequence is far from agreement by different approaches [11,[15][16][17][18][19][20][21][22] Thus, studies of peptides [15,[23][24][25] that model different regions of apoA-I can provide important information on the roles of specific residues and segments, and their interactions in the structure and stability of the parent apolipoprotein. Analysis of the consensus sequences of the A and B repeat derived from the sequences of multiple exchangeable apolipoproteins [7], together with studies of 44-residue consensus sequence peptides, showed ~ 90% and 5 0% α-helical contents for peptides with ABAB [26] and ABBA [27] sequence ordering respectively.…”

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Conformation and Lipid Binding of a C-Terminal (198−243) Peptide of Human Apolipoprotein A-I

Zhu

Atkinson

2007

Biochemistry

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Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on apoA-I demonstrated that this N-terminal region is unstructured and folds into ~ 60% α-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46 residue peptide, apoA-I, encompassing putative helix repeats 10, 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far ultraviolet circular dichroism spectra show that apoA-I is also unfolded in aqueous solution. However, self-association induces ~ 50% α-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid mimicking detergents, above their CMC, ~ 60% α-helix was induced in the peptide. In contrast, SDS, an anionic lipid mimicking detergent, induced helical folding in the peptide at a concentration of ~ 0.003% (1 00 μM), ~ 70 fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solublize dimyristoyl phosphatidyl choline (DMPC) liposomes and fold into ~ 60% α-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy, demonstrated that the peptide binds to DMPC with high affinity to form at least two sizes of relatively homogenous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide w/w, diameter and density) of both complexes are similar to those of plasma A-I/DMPC formed under similar conditions: small discoidal complexes (~ 3:1 w/w, ~ 110Å and ~ 1.10g/cm 3 ) formed at initial 1:1 w/w ratio and larger discoidal complexes ( ~ 4.6:1 w/w, ~ 165 Å and ~ 1.085g/cm 3 ) formed at initial 4:1 w/w ratio. The cross-linking of the peptide on the two sizes of disks is consistent with the calculated peptide numbers per particle, which result in sufficient helix to surround the lipid bilayer twice. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this Cterminal peptide model can mimic the interaction with phospholipid of plasma apoA-I to form two sizes of homogenous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.The plasma concentration of high-density lipoprotein (HDL) is an inverse marker of potential cardiovascular disease. The well-documented anti-atherogenic function of HDL is related to its critical role in reverse cholesterol transport, such as mediation of cholestero...

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Apolipoprotein A‐I peptide models as probes to formulate potential inhibitors of the low‐density lipoprotein oxidation

Darvari

Petraki

Tellis

et al. 2011

Journal of Peptide Science

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Apolipoprotein A-I (apoA-I), which constitutes the principal protein component of high-density lipoprotein, is responsible for its major antiatherogenic functions. Aiming at contributing to the development of potent inhibitors of low-density lipoprotein (LDL) peptide models of helices 4,6 and 9,10 of apoA-I were designed and synthesized. Specific amino acid substitutions, resulting in transformation of the original helix class A and Y to G according to the Schiffer and Edmundson helical wheel representation, were introduced in order to validate the contribution of these modifications in the inhibitory activity of the synthesized peptide models against the LDL oxidation. The role of Met at positions 112 (helix 4) and 148 (helix 6) as oxidant scavenger was also investigated. The helical characteristics of all the peptide models were studied by CD in membrane-mimicking microenvironments and compared with the original helices.

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Probing the Lipid-Free Structure and Stability of Apolipoprotein A-I by Mutation

Cited by 49 publications

References 47 publications

Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

Conformation and Lipid Binding of a C-Terminal (198−243) Peptide of Human Apolipoprotein A-I

Apolipoprotein A‐I peptide models as probes to formulate potential inhibitors of the low‐density lipoprotein oxidation

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