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Illustration of two hypothetical conformations of phospholipid-bound apoA-I. Blue numbered cylinders represent the proline-punctuated eight 22-mers and two 11-mers as sequential repeating units (numbered 1-10) based on computer alignment studies (40). Both models show the relative position of an acceptor and donor probe labeled at position 132 (repeat 5) in the mutant Q132C apoA-I. A, a 104-Å diameter discoidal rHDL particle composed of approximately 185 molecules of DMPC surrounded by two apoA-I monomers, which have been arranged as a pair of continuous amphipathic-helices forming a belt conformation. B, a discoidal rHDL particle of the same size and composition as that shown in the top panel, but where each of the two apoA-I monomers have been arranged to form antiparallel-helices along the edge of the particle forming a picket fence conformation.
Source publication
Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientatio...
Contexts in source publication
Context 1
... Models of Lipid-bound ApoA-I Conformation- Two hypothetical models for the lipid-bound conformation of apoA-I are shown in Fig. 1. Panel A shows the recently pro- posed belt conformation, and panel B shows the picket fence conformation. In the belt conformation, residues at position 132 are approximately 16 Å apart. While in the picket fence con- formation these residues are approximately 104 Å apart. Thus, probe attachment at the repeat 5 position 132 provided ...
Context 2
... that there was no energy transfer when rHDL was disrupted with NaOH/SDS. These data indicate that residues at position 132 in two mono- mers of apoA-I bound to rHDL were close enough to give sub- stantial energy transfer between donor and acceptor probes. This supports the belt model of rHDL in which residues at position 132 are close together (Fig. 1). However, these data by themselves do not rule out models intermediate between the belt and picket fence, because the observed energy transfer efficiency of 40.5% could result from an intermediate spacing between the probes. Such intermediate models were ruled out by analysis of the donor fluorescence lifetime distribution. These data ...
Context 3
... of the lipid-bound conformation of apoA-I has proven to be difficult leading to a recent debate. To distinguish between the two popular models describing the conformation of apoA-I (illustrated in Fig. 1) we have used fluorescence reso- nance energy transfer. In these studies, glutamine 132 within repeat 5 was mutated to cysteine so we could attach thiol- reactive fluorescent donor or acceptor probes at a single defined helical position within the protein. This position was picked for several reasons, first because of the extreme ...
Context 4
... glutamine 132 within repeat 5 was mutated to cysteine so we could attach thiol- reactive fluorescent donor or acceptor probes at a single defined helical position within the protein. This position was picked for several reasons, first because of the extreme separation dis- tance in donor and acceptor probes predicted from each of the two models (Fig. 1). Residues at position 132 would be approx- imately 16 Å from each other in the belt conformation, whereas residues at position 132 would be approximately 104 Å apart in the picket fence conformation. Thus, probe placement at this site allowed each model to serve as its own control. Finally, we chose to attach probes at position 132 ...
Citations
... Une autre utilité de modier des protéines pour qu'elles expriment une paire de uorophore donneur -accepteur est l'étude de leur conformation. Si on souhaite révéler dans quelle conformation une protéine se replie parmi les diérentes conformations possibles, on place un uorophore donneur et un accepteur de telle manière qu'il y aura un niveau important de FRET dans une conformation envisagée, et un FRET nul dans l'autre conformation (gure 3.7) [110]. conformation ceinture (à gauche), il y a FRET entre les 2 uorophores, alors qu'en conformation barrière (à droite) le FRET n'est pas possible, d'après [110] Pour étudier la mécanotransduction, une dernière classe de biosenseurs a été développée. ...
... Si on souhaite révéler dans quelle conformation une protéine se replie parmi les diérentes conformations possibles, on place un uorophore donneur et un accepteur de telle manière qu'il y aura un niveau important de FRET dans une conformation envisagée, et un FRET nul dans l'autre conformation (gure 3.7) [110]. conformation ceinture (à gauche), il y a FRET entre les 2 uorophores, alors qu'en conformation barrière (à droite) le FRET n'est pas possible, d'après [110] Pour étudier la mécanotransduction, une dernière classe de biosenseurs a été développée. Ces biosenseurs permettent d'observer l'activité des molécules de la signalisation cellulaire. ...
Les cellules vivantes sont capables de réagir aux signaux mécaniques tels que la rigidité de la surface sur laquelle elles adhèrent, les forces de tractions ou compressions auxquelles elles sont soumises, le flux de liquide à la surface de leur membrane ou encore la géométrie de leurs adhésions ou de leur forme globale. Ces signaux influent sur des processus cellulaires tels que la prolifération, la différenciation, la migration et la mort cellulaire. Ces processus sont finement régulés par des réactions biochimiques qui forment un réseau de signalisation. La mécanotransduction est la traduction du signal mécanique en signal biochimique.C’est dans le but d’étudier la mécanotransduction que nous avons étudié l’utilisation d’ultrasons pour stimuler mécaniquement les cellules à des fréquences temporelles et spatiales relativement élevées. De nombreux montages expérimentaux et de nombreuses voies ont été considérées dans cette partie très exploratoire. Nous en retenons finalement des pistes prometteuses pour la continuation future de ce projet.Nous avons développé ce que nous nommons des substrats actifs, qui nous permettent de contrôler à la fois spatialement et temporellement la stimulation mécanique appliquée à des cellules vivantes. Ces substrats actifs consistent en des micropiliers de fer incrustés dans un élastomère peu rigide (PDMS) et manipulés par deux électroaimants. Nous pouvons contrôler dynamiquement le déplacement des piliers qui vont déformer localement et de manière continue la surface. Cette déformation va ensuite déformer en traction ou en compression les cellules vivantes étalées sur la surface à proximité. En employant des marqueurs fluorescents nous pouvons réaliser de la Microscopie de Forces de Traction et surveiller la contrainte appliquée par les piliers aux cellules à travers la surface de PDMS, et nous pouvons étudier la réponse mécanique des cellules. De plus, ces substrats sont compatibles avec la microscopie de fluorescence en cellule vivante, ce qui rend possible l’observation de la réponse cellulaire au niveau morphologique (forme des adhésions focales, activité protrusive, …) et surtout biochimique.En effet, pour étudier la réponse biochimique des cellules après une stimulation mécanique, nous observons par microscopie de fluorescence des biosenseurs portant des paires de fluorophores donneur/accepteur. Ces biosenseurs nous permettent d’observer l’activité de protéines impliquées dans la signalisation cellulaire en calculant l’efficacité de Transfert d’Énergie Résonnant de Förster (FRET) de ces biosenseurs. Pour ce faire, les échantillons sont illuminés alternativement aux longueurs d’ondes d’excitation des fluorophores donneurs puis accepteurs. Le signal de fluorescence est collecté simultanément dans un canal d’émission du donneur et un canal d’émission de l’accepteur. Une grande partie de ma thèse a été consacrée à la mise au point d’une méthode quantitative pour analyser les images de fluorescence afin de mesurer une efficacité de FRET qui ne dépende pas de facteurs expérimentaux ni de la quantité de biosenseurs présents dans les cellules. Nous évaluons alors les différentes méthodes pour déterminer les facteurs de correction répandus corrigeant le débordement de spectre du donneur dans le canal accepteur et l’excitation directe de l’accepteur à la longueur d’onde d’excitation du donneur. Pour obtenir des mesures plus quantitatives, nous avons mis au point une nouvelles méthode pour déterminer 2 facteurs de correction supplémentaires. Nous comparons cette méthode à la seule préexistante et évaluons l’influence des paramètres de traitement des images sur les valeurs d’efficacité de FRET mesurées.
... The concept that APOA1 may exist in at least two helical registries is consistent with prior fluorescence energy transfer experiments by Li et al. (30) and raises the intriguing possibility that the shifting of APOA1 helical registries may alter its interactions with HDL remodeling proteins. In the current study, we first confirmed the presence of differing APOA1 helical registries in rHDL, using an orthogonal technique vs. our original cross-linking observation. ...
APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDLs cardioprotective functions, in part via its activation of the enzyme LCAT. On nascent, discoidal HDL, APOA1 comprises 10 alpha-helical repeats arranged in an anti-parallel, stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed Cys-residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1s ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDL that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages - indicating functional particles. However, unlike the 5/5 registry or the wild-type, the 5/2 registry impaired LCAT cholesteryl esterification activity (p<0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and 3-4 on the other. Thus, APOA1 may use a reciprocating, thumbwheel-like mechanism to activate HDL-remodeling proteins.
... The belt model picked up more support with methodologically updated IR experiments performed by Koppaka et al. (1999) that contradicted the earlier IR studies supporting the picket fence in rHDL discs. Over the next few years, several laboratories reported results that supported the belt orientation using methodologies like fluorescence energy transfer (Li et al. 2000) and lipid-based fluorescence quenching (Panagotopulos et al. 2001). With the question of helical orientation largely addressed, much of the focus for the first decade of the 2000s centred on determining the spatial relationships between apoA-I molecules on these discs. ...
Aims:
High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterized.
Methods:
Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.
Results:
rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.
Conclusions:
Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics.
... Crystal structures of truncation mutations of apoA-I, ∆1-43 and ∆185-243, N-and C-terminal deletion mutations, 32,33 respectively, have revealed that the central region prefers to crystallize in a saddle or semicircular conformation, reminiscent of the conformations deduced for apoA-I on discoidal rHDL. [34][35][36][37] If the solution structure of lipid-free apoA-I is compact and highly associated how does it form HDLs? ...
... The basic question is illustrated in Figure 1A showing compact, lipid-free apoA-I and the first product of lipidation, which has the apoA-I belting phospholipid. On the one hand cholate dialysis in the presence of palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) yields rHDL, 9.6 diameter lipid discs carrying about 120 POPC where two antiparallel apoA-I molecules act like a belt around the periphery with the 5,5' amphipathic helices of the two apoA-Is adjacent to one another, [34][35][36]38,39 the LL5/5 rotomer orientation. 40 In the presence of ABCA1, in this case human embryonic kidney 293 (HEK) cells expressing human ABCA1, the predominant nHDL was spherical, had a diameter of from 9-14 nm, carried 109 cholesterol molecules with 130 molecules of phospholipid and three antiparallel molecules of apoA-I that seem to have maintain 5,5' helix association. ...
... 61,64 The purified rHDL was then analyzed on 4-30% non-denaturing gels (CBS Scientific) to assess yield and particle size. 34,36 Cell Culture and Purification of Nascent HDL. HEK 293 cells expressing ABA1 were a generous gift from Dr. Michael Hayden, University of British Columbia, Canada and supplied by Dr. John Parks. ...
The first step in removing cholesterol from a cell is the ATP-binding cassette transporter (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apoA-I, which yields cholesterol-rich nascent HDL (nHDL) that then mature in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3-D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3-D conformation of apoA-I to that of 9-14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. Ten apoA-I mutants were prepared that contained two strategically placed cysteines to form intramolecular disulfide bonds and several that would not form intramolecular disulfide bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was measured from this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with intramolecular disulfide bond present, or unlocked form, cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed on locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation.
... Revealing the structures of biologically active apo A-I in both lipid-free and lipid-bound states are essential in understanding the molecular mechanisms of apo A-I function [6]. Lipidbound apo A-I has been intensively studied by different experimental strategies [7,8], such as X-ray crystallography [9,10], fluorescence resonance energy transfer (FRET) [11,12], small angle neutron scattering [13,14] and chemical cross-linking and mass spectrometry [15][16][17]. Through combining molecular dynamics (MD) simulations with experimental methods researchers have made great progress in refining the three-dimensional structure of apo A-I in lipid-bound states such as discoidal HDL [18][19][20] and spherical HDL [21,22]. ...
Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore, by integrating various experimental results, we proposed a new structural model for lipid-free apo A-I, which contains a bundled four-helix N-terminal domain (1-192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193-243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.
... Finally, these selections were consistent with the structures that docked into the reconstructed envelope. The lipid-binding domain of ApoAI (residues 44-243) consists of a series of eight 22-mer and two 11-mer amphipathic a-helices, which are interrupted by prolines or glycines 39 . To take into account both the high strand flexibility in the regions between the 10 helices as well as the hydrophobic shielding nature of ApoAI* against EmrE, we constructed several alternative models based on the structural framework provided by EOM. ...
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic ‘shield’ that sequesters the IMP from water and promotes its solubilization.
... Finally, these selections were consistent with the structures that docked into the reconstructed envelope. The lipid-binding domain of ApoAI (residues 44-243) consists of a series of eight 22-mer and two 11-mer amphipathic a-helices, which are interrupted by prolines or glycines 39 . To take into account both the high strand flexibility in the regions between the 10 helices as well as the hydrophobic shielding nature of ApoAI* against EmrE, we constructed several alternative models based on the structural framework provided by EOM. ...
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization.
... The belt model picked up more support with methodologically updated IR experiments performed by Koppaka et al. (1999) that contradicted the earlier IR studies supporting the picket fence in rHDL discs. Over the next few years, several laboratories reported results that supported the belt orientation using methodologies like fluorescence energy transfer (Li et al. 2000) and lipid-based fluorescence quenching (Panagotopulos et al. 2001). With the question of helical orientation largely addressed, much of the focus for the first decade of the 2000s centred on determining the spatial relationships between apoA-I molecules on these discs. ...
A molecular understanding of high-density lipoprotein (HDL) will allow a more complete grasp of its interactions with key plasma remodelling factors and with cell-surface proteins that mediate HDL assembly and clearance. However, these particles are notoriously heterogeneous in terms of almost every physical, chemical and biological property. Furthermore, HDL particles have not lent themselves to high-resolution structural study through mainstream techniques like nuclear magnetic resonance and X-ray crystallography; investigators have therefore had to use a series of lower resolution methods to derive a general structural understanding of these enigmatic particles. This chapter reviews current knowledge of the composition, structure and heterogeneity of human plasma HDL. The multifaceted composition of the HDL proteome, the multiple major protein isoforms involving translational and posttranslational modifications, the rapidly expanding knowledge of the HDL lipidome, the highly complex world of HDL subclasses and putative models of HDL particle structure are extensively discussed. A brief history of structural studies of both plasma-derived and recombinant forms of HDL is presented with a focus on detailed structural models that have been derived from a range of techniques spanning mass spectrometry to molecular dynamics.
... http As exchangeable apolipoproteins, both apoAI and apoE3 exhibit the capability to exist in lipid-free and lipoprotein-bound states, with a conformational change accompanying the transition between the two states. Several biophysical approaches indicate that reconstitution of HDL using apoAI and synthetic phospholipids (typically DMPC or POPC) and cholesterol results in the protein adopting a double belt-like organization of a-helices circumscribing a bilayer of phospholipids [5,13,14]. Much less is known about the organization of lipid-associated apoE3 although the available data suggest a similar organization in reconstituted HDL (rHDL) [15][16][17][18]. ...
... Early models agreed that apoA-I was located on the edge of the disk, but disagreed on the conformation of apoA-I (12) (13). After Borhani et al. (7) showed the circular structure for crystalline apoA-I other studies began to confirm this arrangement in a non-crystalline matrix (14)(15)(16)(17)(18)(19). One of the first lipidated apoA-Is studied by chemical cross-linking was synthetic, rHDL (rHDL) prepared from lipid-free apoA-I and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (16,17,20). ...
Apolipoprotein AI (apoA-I) is the principal acceptor of lipids from ATP-binding cassette transporter A1, a process that yields nascent high density lipoproteins. Analysis of lipidated apoA-I conformation yields a belt or twisted belt in which two strands of apoA-I lie antiparallel to one another. In contrast, biophysical studies have suggested that a part of lipid-free apoA-I was arranged in a 4-helix bundle. To understand how lipid-free apoA-I opens from a bundle to a belt while accepting lipid it was necessary to have a more refined model for the conformation of lipid-free apoA-I. This study reports the conformation of lipid-free apoA-I using lysine-to-lysine chemical cross-linking in conjunction with disulfide cross-linking achieved using selective cysteine mutations. After proteolysis cross-linked peptides were verified by sequencing using tandem mass spectrometry. The resulting structure is compact with roughly 4 helical regions, roughly amino acids 44 through 186, bundled together. C- and N-terminal ends, amino acids 1-43 and 187-243, respectively, are folded such that they lie close to one another. An unusual feature of the molecule is the high degree of connectivity of lysine40 with 6 other lysines, lysines that are close, e.g., lysine59, to distant lysines, e.g., lysine239, that are at the opposite end of the primary sequence. These results are compared and contrasted with other reported conformations for lipid-free apoA-I.
