scholarly journals Ligand-induced conformation change in folate-binding protein

1993 ◽  
Vol 292 (3) ◽  
pp. 921-925 ◽  
Author(s):  
N C Kaarsholm ◽  
A M Kolstrup ◽  
S E Danielsen ◽  
J Holm ◽  
S I Hansen
Keyword(s):  
Fluorescence Spectroscopy ◽  
Ligand Binding ◽  
Binding Protein ◽  
Simultaneous Detection ◽  
Tryptophan Fluorescence ◽  
Random Coil ◽  
Folate Binding Protein ◽  
Beta Strand ◽  
Process Detection ◽  
Induced Aggregation

C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cow's whey. The c.d. spectrum of unligated FBP predicts the following secondary structure: 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn and 31% random-coil structure. Folate binding to FBP results in significant changes in the c.d. spectrum. Analysis of the spectrum shows a 10% decrease in antiparallel beta-strand as a result of ligand binding. Folate binding also leads to strong quenching of FBP tryptophan fluorescence. The magnitude of the quench is proportional to ligand binding. The guanidinium chloride-induced unfolding of FBP is shown to be a multistate process. Detection by c.d. and fluorescence spectroscopy lead to non-identical transitions. Modelling studies are consistent with the existence of a stable folding intermediate. Ligand binding to FBP increases the apparent folding stability of the molecule. Simultaneous detection by c.d. and fluorescence indicate that the apparent increased folding stability is derived from ligand-induced aggregation of FBP.

Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

2001 ◽  
Vol 21 (3) ◽  
pp. 305-313 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Ligand Binding ◽  
Binding Protein ◽  
Tryptophan Fluorescence ◽  
Exchange Chromatography ◽  
Cow’S Milk ◽  
Conformation Change ◽  
Ionic Charge ◽  
Folate Binding Protein ◽  
Cow's Milk ◽  
Isoelectric Points

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.

Binding of Radiolabeled Folate and 5-Methyltetrahydrofolate to Cow's Milk Folate Binding Protein at pH 7.4 and 5.0. Relationship to Concentration and Polymerization Equilibrium of the Purified Protein

2001 ◽  
Vol 21 (6) ◽  
pp. 733-743 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Conformational Changes ◽  
Protein Conformation ◽  
Binding Protein ◽  
Cow’S Milk ◽  
Folate Binding Protein ◽  
Cow's Milk ◽  
Polymerization Equilibrium ◽  
Methyl Tetrahydrofolate

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 μM) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.

scholarly journals The role of glycosylation in the biosynthesis and acquisition of ligand- binding activity of the folate-binding protein in cultured KB cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1171-1180 ◽  
Author(s):  
CA Luhrs
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Property ◽  
Affinity Matrix ◽  
Kb Cells ◽  
Folate Binding Protein ◽  
Binding Function ◽  
High Mannose ◽  
Endoglycosidase H

Abstract The biosynthesis, processing, and ligand-binding function of the membrane-associated and soluble forms of the folate-binding protein (FBP) in KB cells, a cultured human cell line, were studied using pulse- chase labeling with [35S] methionine. The intermediary and mature forms of the protein were isolated by immunoprecipitation and affinity chromatography and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography. The earliest species identified had an Mr of 32 Kd and disappeared over 5 hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd species disappeared, a 40-Kd form appeared in the medium. When tunicamycin was added to the culture medium to inhibit core glycosylation, a 26-Kd aglycosylated species and minor 28- Kd and 30-Kd forms appeared. Endoglycosidase H, which cleaves high mannose but not complex oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no effect on the 38-Kd form, indicating that this species is complex glycosylated. Monensin, which blocks complex glycosylation, also inhibited synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had ligand-binding sites (as demonstrated by binding to a folate-Sepharose matrix), the 26-Kd aglycosylated species, labeled in the presence of tunicamycin, lacked similar binding sites because it did not bind to the affinity matrix. In contrast, the aglycosylated 26-Kd form, which was obtained by treatment of the 32-Kd species with endoglycosidase H, did bind to the folate affinity matrix, indicating that it retained ligand-binding function. Thus, the high mannose oligosaccharide moiety is not required for the folate-binding property of the FBP, but its addition to the polypeptide chain precedes a later step that is necessary for the mature protein to have ligand-binding function.

scholarly journals The role of glycosylation in the biosynthesis and acquisition of ligand- binding activity of the folate-binding protein in cultured KB cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1171-1180
Author(s):  
CA Luhrs
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Property ◽  
Affinity Matrix ◽  
Kb Cells ◽  
Folate Binding Protein ◽  
Binding Function ◽  
High Mannose ◽  
Endoglycosidase H

The biosynthesis, processing, and ligand-binding function of the membrane-associated and soluble forms of the folate-binding protein (FBP) in KB cells, a cultured human cell line, were studied using pulse- chase labeling with [35S] methionine. The intermediary and mature forms of the protein were isolated by immunoprecipitation and affinity chromatography and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography. The earliest species identified had an Mr of 32 Kd and disappeared over 5 hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd species disappeared, a 40-Kd form appeared in the medium. When tunicamycin was added to the culture medium to inhibit core glycosylation, a 26-Kd aglycosylated species and minor 28- Kd and 30-Kd forms appeared. Endoglycosidase H, which cleaves high mannose but not complex oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no effect on the 38-Kd form, indicating that this species is complex glycosylated. Monensin, which blocks complex glycosylation, also inhibited synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had ligand-binding sites (as demonstrated by binding to a folate-Sepharose matrix), the 26-Kd aglycosylated species, labeled in the presence of tunicamycin, lacked similar binding sites because it did not bind to the affinity matrix. In contrast, the aglycosylated 26-Kd form, which was obtained by treatment of the 32-Kd species with endoglycosidase H, did bind to the folate affinity matrix, indicating that it retained ligand-binding function. Thus, the high mannose oligosaccharide moiety is not required for the folate-binding property of the FBP, but its addition to the polypeptide chain precedes a later step that is necessary for the mature protein to have ligand-binding function.

The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction

2011 ◽  
Vol 1810 (12) ◽  
pp. 1330-1339 ◽  
Author(s):  
Jan Holm ◽  
Christian Schou ◽  
Linnea N. Babol ◽  
Anders J. Lawaetz ◽  
Susanne W. Bruun ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
Self Association

Effect of Hydrogen Ion Concentration and Buffer Composition on Ligand Binding Characteristics and Polymerization of Cow's Milk Folate Binding Protein

2001 ◽  
Vol 21 (6) ◽  
pp. 745-753 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Ligand Binding ◽  
Binding Protein ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Cow’S Milk ◽  
Folate Binding Protein ◽  
Hydrogen Ion Concentration ◽  
Binding Characteristics ◽  
Cow's Milk ◽  
Buffer Composition

The ligand binding and aggregation behavior of cow's milk folate binding protein depends on hydrogen ion concentration and buffer composition. At pH 5.0, the protein polymerizes in Tris-HCl subsequent to ligand binding. No polymerization occurs in acetate, and binding is markedly weaker in acetate or citrate buffers as compared to Tris-HCl. Polymerization of ligand-bound protein was far more pronounced at pH 7.4 as compared to pH 5.0 regardless of buffer composition. Binding affinity increased with decreasing concentration of protein both at pH 7.4 and 5.0. At pH 5.0 this effect seemed to level off at a protein concentration of 10−6 M which is 100–1000 fold higher than at pH 7.4. The data can be interpreted in terms of complex models for ligand binding systems polymerizing both in the absence or presence of ligand (pH 7.4) as well as only subsequent to ligand binding (pH 5.0).

scholarly journals HUMAN SERUM FOLATE CAN BE MEASURED USING FOLATE BINDING PROTEIN LINKED TO ENZYME-LABELED PROTEIN LIGAND BINDING ASSAY (ELPLBA) AS WELL AS ELISA

2019 ◽  
Vol 1 (2) ◽  
pp. 59-67
Author(s):  
Muhamad Arif Budiman ◽  
Mohamad Sadikin ◽  
Ani Retno Prijanti
Keyword(s):  
Ligand Binding ◽  
Binding Assay ◽  
Binding Protein ◽  
Serum Folate ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ligand Binding Assay ◽  
Elisa Method ◽  
Folate Binding Protein ◽  
Significant Difference

Background: Folate is an important substance used for purine and pyrimidine nucleotide synthesis. One measurement of folate that already establishes is using ELISA (Enzyme-linked immunosorbent assay) method. Folate binding protein is a protein that can bind folate, therefore it considered can be used as a tool that can replace antibody dependent ELISA method.Objectives: The aim of this research was to create a method for folate measurement in serum called Enzyme-labeled protein ligand binding assay (ELPLBA) by replacing antibody as used in ELISA method with folate binding protein (FBP) that purified from the whey of milk.Methods: The method is tested using 20 serum samples and compared to ELISA. Folate binding protein was purified from bovine’s milk using ammonium sulfate up to 90% saturated, DEAE-cellulose anion exchange chromatography and affinity chromatography. SDS-PAGE and western blot were used to establish the protein band of FBP that has molecular weight of ~25-35 kDa. ELPLBA was arranged with stationary phase using aminohexyl-agarose, and folic acid linked on it using carbodiimide.Results: The result show there was no significant difference of folate concentration between ELPLBA (14.804 ± 2.795) and ELISA method (13.859 ± 3.638), p = 0.363.Conclusion:  ELPLBA method show similarity for determination of folate in serum which was the same as standard folate measurement (ELISA).

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