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
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.
Available from: Marco Rito-Palomares
- "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. "
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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 · 2.15 Impact Factor
Available from: Antonio Diaz
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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
Available from: Neelesh Mehra
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ABSTRACT: Receptor-based targeting of therapeutics may be a fascinating proposition to improve the therapeutic efficacy of encapsulated drugs. The development of safe and effective nanomedicines is a prerequisite in the current nanotechnological scenario. Currently, the surface engineering of nanocarriers has attracted great attention for targeted therapeutic delivery by selective binding of targeting ligand to the specific receptors present on the surface of cells. In this review, we have discussed the current status of various receptors such as transferrin, lectoferrin, lectin, folate, human EGF receptor, scavenger, nuclear and integrin, which are over-expressed on the surface of cancer cells; along with the relevance of targeted delivery systems such as nanoparticles, polymersomes, dendrimers, liposomes and carbon nanotubes. The review also focuses on the effective utilization of receptor-based targeted delivery systems for the management of cancer in effective ways by minimizing the drug-associated side effects and improving the therapeutic efficacy of developed nano-architectures.
Therapeutic delivery 03/2013; 4(3):369-94. DOI:10.4155/tde.13.6
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