A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

Velkov, Tony; Lim, Maria L. R.; Capuano, Ben; Prankerd, Richard J.

doi:10.1016/j.jchromb.2008.04.011

Cited by 13 publications

(15 citation statements)

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“…4A–C, insets and SI Table 1). In order to examine the effect of endogenous lipidic substances that remain bound to AGP throughout the purification process [8], a sample of AGP was subjected to a delipidation process [45] and compared to a sample from the same preparation for PmB binding at 25 °C (Fig. 4F and SI Table 1).…”

Section: Resultsmentioning

confidence: 99%

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Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein

Azad¹,

Huang

Cooper

et al. 2012

Biochemical Pharmacology

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Here, for the first time, we have characterized binding properties of the polymyxin class of antibiotics for human α-1-acid glycoprotein (AGP) using a combination of biophysical techniques. The binding affinity of colistin, polymyxin B, polymyxin B3, colistin methansulfonate, and colistin nona-peptide was determined by isothermal titration calorimetry (ITC), surface plasma resonance (SPR) and fluorometric assay methods. All assay techniques indicated colistin, polymyxin B and polymyxin B3 display a moderate binding affinity for AGP. ITC and SPR showed there was no detectable binding affinity for colistin methansulfonate and colistin nona-peptide, suggesting both the positive charges of the diaminobutyric acid (Dab) side chains and the N-terminal fatty acyl chain of the polymyxin molecule are required to drive binding to AGP. In addition, the ITC and fluorometric data suggested that endogenous lipidic substances bound to AGP provide part of the polymyxin binding surface. A molecular model of the polymyxin B3–AGP F1*S complex was presented that illustrates the pivotal role of the N-terminal fatty acyl chain and the D-Phe6-L-Leu7 hydrophobic motif of polymyxin B3 for binding to the cleft-like ligand binding cavity of AGP F1*S variant. The model conforms with the entropy driven binding interaction characterized by ITC which suggests hydrophobic interactions coupled to desolvation events and conformational changes are the primary driving force for polymyxins binding to AGP. Collectively, the data are consistent with a role of this acute-phase reactant protein in the transport of polymyxins in plasma.

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Section: Resultsmentioning

confidence: 99%

“…Delipidation of AGP was performed as previously reported [45] using lipidex 1000 (a hydroxyalkylpropyl (HAP) derivative of Sephadex G25 substituted 10% with alkyl chains of C 15 –C 18 in length syn. (HAP)-dextran type VI syn.…”

Section: Methodsmentioning

confidence: 99%

Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein

Azad¹,

Huang

Cooper

et al. 2012

Biochemical Pharmacology

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“…no. 17-1085-01) as previously described [54]. The final purity of the proteins was ascertained by SDS-PAGE (silver staining) and in all cases was >98% (Figure 1).…”

Section: Methodsmentioning

confidence: 99%

Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

Velkov

2013

PPAR Research

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Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD) of PPARα. This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPARαLBD. The role of L-FABP in the delivery of ligands directly to PPARα via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed.

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“…Delipidation of LFABP was performed by the protocol of Velkov et al (26). Briefly, bacterial cells were lysed by sonication in a buffer containing protease inhibitors and 1 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

Fatty Acid Induced Remodeling within the Human Liver Fatty Acid-binding Protein

Sharma

2011

Journal of Biological Chemistry

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We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP.Liver fatty acid-binding protein (LFABP) 2 is an intracellular lipid chaperone belonging to a family of ϳ15 kDa intracellular lipid-binding proteins (iLFABP). LFABP, along with the other iLBP family members, appears to maintain physiologically relevant concentrations of cytosolic fatty acids (1-6). Increased expression of LFABP in humans is associated with insulin-dependent diabetes and gestational diabetes (7-9). Studies with LFABP gene-ablated mice demonstrate a physiological role for LFABP in hepatic fatty acid metabolism and in diet-induced obesity (10 -12). Mechanistic details of LFABP-fatty acid interaction have not as yet resolved the mode of binding for both fatty acid molecules. This is in part due to the lack of high resolution crystal structures of ligand-free and ligand-bound forms of human LFABP. Unlike other members of the iLFABP family, LFABP is unique in binding two fatty acid molecules (13,14). It can also bind bile salts, acyl-CoA esters, and other hydrophobic compounds (15)(16)(17)(18)(19). LFABP shares a common structural motif with other iFABPs comprising a 10 -11-stranded ␤ barrel that forms a ligand binding cavity, which is covered by a helix-turn-helix (HTH) motif (20). This HTH along with neighboring ␤ turns between ␤ strands is hypothesized to form a portal to the protein cavity, allowing ligand trafficking without significant structural rearrangements in the ␤ barrel (21-24). The terminology of apo-and holo-used in this work refers to ligand-bound and ligand-unbound states of FABP, and this terminology is used here to remain consistent with previously published works on FABP (21). We crystallized the human LFABP in apo, holo, and in intermediate states of fatty acid (palmitate) binding to obtain structural snapshots of fatty acid engagement, entry, and final docking within LFABP. Our data provide crystallographic evidence for hitherto unexplored heads-in modes of entry for fatty acids in LFABP. The analyses are supported by earlier mutagenesis experiments (13), and together these data reveal the atomic reconstruction required within LFABP to accommodate two chains of fatty acids. Targeting the newly identified critical residues within LFABP, which undergo conformational alterations to accommodate two fatty acids, with small molecule inhibitors may be a potent new strategy for developing anti-FABP drugs. EXPERIMENTAL PROCEDURESOverexpression and Purification of LFABP-The full-length human LFABP was cloned and overexpressed as described previously (25). Delipidation of LFABP was performed by ...

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A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

Cited by 13 publications

References 46 publications

Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein

Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein

Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

Fatty Acid Induced Remodeling within the Human Liver Fatty Acid-binding Protein

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