Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein
ABSTRACT High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 nm to 460-470 nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements.
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- "ANS is an extrinsic fluorescent probe that emits large fluorescence energy at 470 nm when anilinonaphthalene group binds to hydrophobic areas of proteins. ANS has been widely used in numerous investigations to study hydrophobic interactions of proteins (Holm et al., 2012), refolding of proteins (Gautam et al., 2012), protein–ligand interactions (Ding et al., 2010; Tutar et al., 2010), protein aggregation (Bolognesi et al., 2010), protein denaturation (Yadav and Prakash, 2009), protein surface hydrophobicity (Alizadeh-Pasdar and Li-Chan, 2000), and others. Hence, ANS method can be used to quantify superficial hydrophobicity of proteins, previously separated in HIC, in order to analyze its chromatographic behavior when the effect of different parameters in HIC are being studied. "
ABSTRACT: Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity-as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S0 ) for each fraction; a unique correlation between S0 and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how-and in which extent-the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies. Copyright © 2013 John Wiley & Sons, Ltd.Journal of Molecular Recognition 12/2013; 26(12):618-26. DOI:10.1002/jmr.2302 · 2.34 Impact Factor
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ABSTRACT: Background: Crystallization is not always achieved for all proteins in a good size and a good quality for X-ray diffraction. So that condition opens a field for the development of theoretical molecular and protein studies allowing the representation of the molecules in 3D, providing spatial information to study the interaction between ligands and macromolecular receptors. Materials and Methods: In silico study from primary sequence analysis of six different proteins LuxS crystallized of several bacteria. 1J6X protein of Helicobacter pylori was selected for its similarity with the LuxS protein sequence in Porphyromonas gingivalis (P. gingivalis) strain W83 to produce a homology model of this protein, using the Sybyl and MOE software. A docking was performed to assess the reproducibility of the model in a biological environment. Results: The LuxS protein modelling of P. gingivalis strain W83 was developed, which allows the approach to a proposed structure for the interaction between the protein and its natural ligand. The model generated with computational resources achieved the correct position and biological behavior by means of developed calculations. The docking showed a cavity in which the ligand adopted several positions with good results. Conclusions: A LuxS protein model was obtained, validated by different methods. This generated a 3D model for LuxS protein in P. gingivalis strain W83 with biological reproducibility by means of molecular docking.12/2012; 5(3):105-113. DOI:10.4067/S0719-01072012000300001
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ABSTRACT: The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH7.4 (pI=7-8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3μM holo-FBP showed Tm (76 (0)C) and molar enthalpy (146kcalM(-1)) values increasing to 78 (0)C and 163kcalM(-1) at 10μM holo-FBP, while those of apo-FBP were 55 (0)C and 105kcalM(-1). Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10μM) in DLS after a step-wise rise in temperature to 78 (0)C≈Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH5 (Tm 38 (0)C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.Biochimica et Biophysica Acta 12/2013; 1844(3). DOI:10.1016/j.bbapap.2013.12.009 · 4.66 Impact Factor