One-step purification of rabbit histidine rich glycoprotein by dye-ligand affinity chromatography with metal ion requirement

A simple method for purification of the histidine rich glycoprotein (rHRG) from rabbit sera was developed. The rHRG was purified by one-step affinity chromatography using the triphenylmethane dye "acid fuchsin" as a specific ligand, which gave an overall yield above 80%. Interestingly, the binding of rHRG to the ligand required the divalent transition-metal ions such as Zn2+, Ni2+, and Co2+ at pH 9.5. In the presence of 0.5 mM ZnCl2, the binding was enhanced 15 times compared with that in the absence of ZnCl2. Bound rHRG was efficiently eluted from the affinity absorbent with 100 mM imidazole or histidine. Purified rHRG was homogeneous with an Mr of 94 kDa when analyzed by SDS-PAGE, whereas isoelectric focusing revealed microheterogeniety with pI values ranging from 6.3 to 6.8. Blotting analysis with lectins specific for carbohydrate moieties and treatment with glycosidases demonstrated that rHRG is a highly N-glycosylated protein with diverse carbohydrate structures.     

A single amino acid in VP2 is critical for the attachment of infectious bursal disease subviral particles to immobilized metal ions and DF-1 cells

VP2 protein is the primary host-protective immunogen of infectious bursal disease virus (IBDV). His249 and His253 are two surface histidine residues in IBDV subviral particles (SVP), which is formed by twenty VP2 trimers when the VP2 protein of a local isolate is expressed. Here, a systemic study was performed to investigate His249 or/and His253 on self-assembly, cell attachment and immunogenicity of SVP. Point-mutagenesis of either or both histidine residues to alanine did not affect self-assembly of the SVP, but the SVP lost its Ni-NTA binding affinity when the His253 was mutated. Indirect immunofluorescence assays and inhibitory experiments also showed that His253 is essential for SVP to attach onto the DF-1 cells and to inhibit IBDV infection of DF-1 cells. Finally, enzyme-linked immunosorbent assays and chicken protection assays demonstrated that SVP with a mutation of His253 to alanine induced comparable neutralizing antibody titers in chickens as the wild-type SVP did. It was concluded that VP2's His253, a site not significant for the overall immunogenicity induced by SVP, is crucial for the binding affinity of SVP to Ni-NTA and the attachment of an IBDV host cell line. This is the first paper to decipher the role of His253 played in receptor interaction and immunogenicity.     

Evidence for the participation of histidine residues located in the 56 kDa C-terminal polypeptide domain of ADP-ribosyl transferase in its catalytic activity

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl-terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP-ribosylation.     

Nonexistence of Exo-cellobiohydrolase (CBH) in the Cellulase System of Trichoderma viride

Chemical modification of histidine residues in ricin E was studied with regard to saccharide binding. The analytical data indicate that 6 out of 7 histidine residues in ricin E are eventually modified with diethylpyrocarbonate (DEP) at pH 6.0 and 25°C in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutinating activity of ricin E, and only 10% of the residual activity was found after modification of 6 histidine residues/mol. The data of affinity chromatography using lactamyl- and galactosamine-cellulofine columns suggest that modification of histidine residues does not have much effect on the binding ability at the low affinity saccharide-binding site of ricin E but abolishes the binding ability at the high affinity saccharide-binding site. In the presence of lactose, one histidine residue/mol was protected from the DEP modification with retention of a fairly high cytoagglutinating activity. Such a protective effect was also observed for specific saccharides such as galactose and A^-acetylgalactosamine, but not for glucose, a non-specific saccharide. On treatment with hydroxylamine, the modified ricin E restored 67 % of the cytoagglutinating activity. Based on these findings, it is suggested that in the high affinity saccharide- binding site of ricin E there exists one histidine residue responsible for saccharide binding.     

Development and characterization of Ni-NTA-bearing microspheres

BACKGROUND: For ease of purification, proteins are often expressed with a short affinity sequence of five or six adjacent histidine residues (His-tag). This His-tag binds to the metal of metal chelator complexes such as Ni(2+)-nitrilotriacetic acid (Ni-NTA) or -iminodiacetic acid (Ni-IDA). Chromatography resins bearing covalently attached metal chelator complexes are used widely for the easy affinity purification of His-tagged proteins or peptides. Because Ni-NTA microspheres were not commercially available at the beginning of our studies, we prepared and characterized such microspheres to immobilize His-tagged proteins and study their interactions. Our microspheres are of three types: (a) metal chelator complexes bound covalently to polystyrene microspheres, (b) metal chelator complexes bound covalently to silica microspheres, and (c) lipid-linked metal chelator complexes adsorbed to silica microspheres forming self-assembled bilayer membranes where the metal chelators have lateral mobility. METHODS: The microspheres bearing covalently attached Ni-chelator were synthesized by reacting a primary amine-bearing Ni-NTA ligand with carboxy-functionalized microspheres and then loading with Ni(2+). Microspheres with laterally mobile metal chelator were made by incubating glass microspheres with liposomes containing phosphatidylcholine (PC) and the metal chelating lipid 1,2-dioleoyl-sn-glycero-3-[(N (5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]. Binding of a His-tagged enhanced green fluorescent protein (EGFP) was used to characterize these microspheres by flow cytometry for their specificity, sensitivity, capacity and stability. RESULTS: While all micospheres specifically bind His-tagged proteins, the conditions to achieve this are different for the polystyrene- and silica-based spheres. All three types of microspheres bind His-EGFP with saturation occurring at 30-50 nM and an apparent avidity (concentration of half-maximal binding) of approximately 1 to 2 x 10(-8) M at pH 7.4. Binding of His-EGFP is inhibited by imidazole or ethylene-diaminetetraacetic acid (EDTA). Polystyrene Ni-NTA microspheres showed significant nonspecific binding as measured by binding in the presence of imidazole or EDTA or by binding of fluorescent proteins lacking a His-tag. This nonspecific binding of proteins to and aggregation of polystyrene spheres could only be prevented by the inclusion of low concentrations of Tween 20, but not by including bovine serum albumin (BSA), polyethylene glycols, or polyvinylpyrrolidones as blocking agents. In contrast, silica-based microspheres with covalently attached Ni-NTA or silica microspheres bearing adsorbed bilayers that contain Ni-NTA-lipid showed little nonspecific binding in the presence of BSA. Our results on the stability of immobilization indicate that washing destabilizes the binding of His-tagged proteins to Ni-NTA microspheres. This binding consists of two interactions of different affinities. We also demonstrate that limited multiplexed analysis with differently sized silica microspheres bearing the Ni-NTA-lipid is feasible. CONCLUSIONS: The microspheres described are well suited to selectively immobilize His-tagged proteins to analyze their interactions by flow cytometry. The affinity and kinetic stability of the interaction of His-tagged proteins with Ni-NTA are insufficient to use Ni-NTA microspheres in multiplexed analysis formats where different His-tagged proteins are bound to distinct microspheres. Improvements towards this end (improved chelators and/or improved affinity tags) are critical for extending the use of this method. We are currently working on novel chelators to strengthen the stability of immobilization of His-tagged proteins to surfaces. Such improvements would greatly enhance the analysis of interactions of immobilized His-tagged proteins and could make the development of microsphere-based arrays with His-tagged protein/antibody possible.     

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.     

Ethoxyformylation of bovine growth hormone

Reactivity of histidines in bovine growth hormone towards ethoxyformic anhydride was investigated and localization in the molecule of two kinetically distinguishable classes was achieved, a slow class including only histidine residue 169 (k = 0.180 min-1) and a fast one composed of histidines 19 and 21 (k = 0.900 min-1). Total ethoxyformylation of bovine growth hormone brought about a complete loss of its capacity to compete with 125I-labelled hormone for rat-liver binding sites, but modification of approximately half of the fast histidine group was enough to produce an important decrease in this capacity. Circular dichroism studies indicated no significant changes in protein conformation with all three histidine residues modified. Practically full binding capacity was restored when these residues were regenerated by treatment with hydroxylamine. These results suggest that one or both of the fast reacting histidine residues are involved in bovine growth hormone binding to its specific receptors.     

Serum inhibitors of desialylated glycoprotein binding to hepatocyte membranes.

Identification of the material present in human serum which is responsible for inhibition of binding of desialylated glycoproteins to rat hepatocyte membranes was accomplished by means of affinity chromatography using Sephadex to which the galactose-specific lectin, Ricinus Communis Agglutinin (RCAI) was covalently bound. RCAI-Sephadex was capable of extraction of virtually all of the inhibitory activity from cirrhotic serum. The RCA I-bound inhibitory activity could be eluted with 0.05 M D-galactose. The D-galactose eluate when subjected to radioimmunoelectrophoresis against a number of specific antibodies to human serum glycoproteins produced arcs corresponding to alpha 1-acid glycoprotein, alpha2-macroglobulin, IgG, IgA, and IgM. In another experiment putative terminal galactosyl groups of desialylated glycoproteins in the D-galactose eluate from cirrhotic serum exposed to RCAI-Sephadex were labelled with tritiated borohydride after treatment with galactose oxidase. Subsequent gel electrophoresis showed peaks of radioactivity throughout the area of the gel corresponding to protein molecular weights of the 19 S, 7 S, and 4 S classes. It thus appears that a heterogeneous population of desialylated serum glycoproteins accounts for the inhibition of binding of desialylated glycoprotein to the hepatocyte membrane and that these desialylated glycoproteins are present in small amounts in normal human serum and in greatly increased quantities in serum from patients with cirrhosis.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Fragment length influences affinity for Cu2+ and Ni2+ binding to His96 or His111 of the prion protein and spectroscopic evidence for a multiple histidine binding only at low pH

The prion protein (PrP) is a Cu2+-binding cell-surface glycoprotein. Using various PrP fragments and spectroscopic techniques, we show that two Cu2+ ions bind to a region between residues 90 and 126. This region incorporates the neurotoxic portion of PrP, vital for prion propagation in transmissible spongiform encephalopathies. Pentapeptides PrP-(92-96) and PrP-(107-111) represent the minimum motif for Cu2+ binding to the PrP-(90-126) fragment. Consequently, we were surprised that the appearance of the visible CD spectra for two fragments of PrP, residues 90-126 and 91-115, are very different. We have shown that these differences do not arise from a change in the co-ordination geometry within the two fragments; rather, there is a change in the relative preference for the two binding sites centred at His111 and His96. These preferences are metal-, pH- and chain-length dependent. CD indicates that Cu2+ initially fills the site at His111 within the PrP-(90-126) fragment. The pH-dependence of the Cu2+ co-ordination is studied using EPR, visible CD and absorption spectroscopy. We present evidence that, at low pH (5.5) and sub-stoichiometric amounts of Cu2+, a multiple histidine complex forms, but, at neutral pH, Cu2+ binds to individual histidine residues. We have shown that changes in pH and levels of extracellular Cu2+ will affect the co-ordination mode, which has implications for the affinity, folding and redox properties of Cu-PrP.     

The role of lysine, histidine and carboxyl residues in biological activity of equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L.

Equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L. was subjected to chemical modifications with group specific reagents. Lysine residues were modified with pyridoxal-5'-phosphate, histidine residues with diethyl pyrocarbonate and carboxyl groups with the use of a water soluble carbodiimide. Modification of charged residues had no significant influence on the toxin interaction with serum lipoproteins. Lysine 5'-phosphopyridoxylated and histidine carbethoxylated derivatives of the toxin retained lethal and hemolytic activities, but the pH profile of hemolytic activity of 5'-phospho-pyridoxylequinatoxin II was markedly altered. Modification of the toxin carboxyl groups impaired both hemolytic and lethal activities, the latter, however, to the greater extent.     

Evaluation of the specificity of lectin binding to sections of plant tissue

Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissues. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus and Ulex lectins for L-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (gamma-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.     

A heme- and metal-binding hexapeptide from the sequence of rabbit plasma histidine-rich glycoprotein

Rabbit histidine-rich glycoprotein (HRG) binds low-spin heme and metals tightly at several sites that contain histidine. As part of an on-going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2-Gly-His-Phe-Pro-Phe-His-Trp-... was found in a 16 kDa heme-binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C-terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis-histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not affect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 +/- 25 microM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 microM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60 mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis-histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme- and metal-binding site of rabbit HRG.     

Use of radioactive glucosamine in the perfused rat liver to prepare alpha 1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues.

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.     

The histidine-rich glycoprotein of serum has a domain rich in histidine, proline, and glycine that binds heme and metals

Histidine-rich glycoprotein (HRG) from rabbit serum was digested with plasmin, reduced, and carboxymethylated, and the fragments produced were resolved by reverse-phase high-performance liquid chromatography. Several peptide fractions were obtained that contain unusually high contents of histidine, proline, and glycine. One His-Pro-Gly-rich peptide (apparent Mr 30 000) was obtained in sufficient yield and purity for further study. This peptide is 29 mol % histidine, 37% proline, and 16% glycine, indicating that most of these three amino acids are located in one region of HRG. The peptide contains 9% by weight carbohydrate and is devoid of tyrosine or tryptophan. The far-ultraviolet circular dichroism spectrum of the peptide has a minimum at 203 nm, indicating that the peptide contains polyproline II helical sections. The peptide represents a binding domain of HRG since it retains much of the ability of intact HRG to bind heme and metals including Zn2+, Ni2+, and Cu2+. As with the parent HRG molecule, interaction of the peptide with heme and metals is dependent on pH and intact histidine residues.     

Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.     

Signal Peptide does not Inhibit Binding of Biotin to Streptavidin

Three recombinant polypeptides of streptavidin: the full-length streptavidin with a signal peptide (rsavS), full-length streptavidin (rsavF) and core streptavidin (rsavC), were expressed in E. coli strain BL21 (DE3) and purified by Ni-NTA chromatography. Although all three recombinant streptavidins had biotin-binding activity, the stability and solubility of rsavC tetraunits were much better than those of rsavS and rsavF, indicating that signal peptide and/or extra amino acid residues in rsavS and rsavF have negative effects on streptavidin. Meanwhile, the signal peptide and extra amino acid residues in rsavS and rsavF made it difficult for polypeptides to fold into functional proteins. After refolding of denaturing-purified proteins in vitro, both the specific activities and biotin binding sites of renatured streptavidins were 1.4-times as that of proteins obtained by native Ni-NTA purification. Because the denaturing-purified rsavC is easy of refolding into functional protein, the better strategy for production of active rsavC is to isolate the protein from IPTG-induced E. coli extracts by denaturing Ni-NTA affinity chromatography followed by refolding of purified polypeptide in vitro.     

Homology modelling and H NMR studies of human leukaemia inhibitory factor

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with ¹H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pK_a values of which range from 3.6 to 7.4. These pK_a values were assigned to individual histidine residues from NMR studies on a series of His → Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.     

Diethylpyrocarbonate, a histidine selective reagent, inhibits progestin binding to chick oviduct cytosol

In this report we describe experiments showing that diethylpyrocarbonate, a histidine selective reagent, inhibits progestin binding to the chick oviduct progesterone receptor. Because this inhibition is reversed by hydroxylamine, we suggest that the chick oviduct progesterone receptor contains one or more histidine residues that regulate progestin binding. We also find that the progestin R5020 protects the progesterone receptor from diethylpyrocarbonate mediated inhibition of progestin binding. From this we infer that the progestin binding site contains a histidine residue(s) important for progesterone binding to its receptor in chick oviduct.     

Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis

Regulation of violaxanthin de-epoxidase (VDE) involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. The role of histidine residues in this process was studied by release of unbound enzyme from thylakoids upon sonication, on a pH scale from 4.7 to 7.1. The co-operativity for binding of spinach VDE (four histidines) to the membrane was found to be 3.8, with respect to protons, and had an inflexion point at pH 6.6, whereas VDE from wheat (three histidines) showed a co-operativity of 2.9 and had an inflexion point at pH 6.2. Mutant forms of VDE were constructed and probed for their binding to the outside of thylakoid membranes. With one or two histidines substituted for alanine or arginine, a lower co-operativity (1.6–2.3) was found, compared with the wild type. Based on these findings, and that the pKa value for histidine is within the range where the VDE binding takes place, we propose that protonation of the histidine residues at low pH induces the conformational change of VDE, and hence indirectly regulates binding of the enzyme to the thylakoid membrane.