Electrostatic properties of bovine ?-lactoglobulin

Fogolari, Federico; Ragona, Laura; Licciardi, Stefania; Romagnoli, Silvia; Michelutti, Roberta; Ugolini, Raffaella; Molinari, Henríette

doi:10.1002/(sici)1097-0134(20000601)39:4<317::aid-prot50>3.0.co;2-w

Cited by 79 publications

(85 citation statements)

References 45 publications

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“…The low pK a value (1.05) estimated for Asp 98 in lacb_bovin is in good agreement with the value of 2 that was determined experimentally by NMR (20).…”

Section: Fig 2 Grid Docking Solutions Obtained For Blg (A) and Plg (B)supporting

confidence: 88%

“…Partial atomic charges and radii for the protein were taken from the pK a files provided by UHBD. The electrostatic potential computed by UHBD has been used for generating an ensemble of protonation states at different pHs using a Montecarlo procedure as described previously (20). A straightforward application of this procedure led to few but very large pK a shifts, which were associated with atoms (and therefore with charges) too close to each other.…”

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confidence: 99%

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EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins

Ragona

Fogolari

Catalano

et al. 2003

Journal of Biological Chemistry

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␤-Lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of ␤-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of ␤-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu 89 protonation. The alkaline shift observed for Glu 89 pK a in porcine ␤-lactoglobulin (pK a 9.7) with respect to the bovine species (pK a 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine ␤-lactoglobulin seems to be functional to lipases activity. Theoretical pK a calculations extended to representative ␤-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all ␤-lactoglobulins.The physicochemical and biological characteristics of ␤-lactoglobulins, which belong to the lipocalin family, have been extensively studied in the last 30 years, but despite the wealth of data, the biological function of these extracellular proteins is still undefined (Ref. 1 and references therein). ␤-Lactoglobulins isolated from cow, goat, and sheep milk samples, under nondenaturing conditions, showed endogenously bound fatty acids (2). Many authors have suggested that bovine ␤-lactoglobulin (BLG) 1 has a transport and/or protective role toward bound ligands in the stomach (3). However, we have previously shown (4) that BLG, despite its high stability at acidic pH, is unable to bind fatty acids at low pH, thus indicating that it could not be employed "as is" as a transporter through the human gastric tract.Retinoids and fatty acids have been reported to bind to BLG in vitro in the pH range of 6.5-8.5, with dissociation constants on the order of 65 nM and 0.6 M, respectively (5). Specifically, titration experiments of BLG with palmitic acid (PA) (4) have clearly shown that: (i) at neutral pH the primary site for palmitic acid binding is within the protein calyx; (ii) the amount of bound PA is drastically reduced upon decreasing pH and the ligand is completely released at pH 2; (iii) in the pH range 7.3-6.4, a conformational equilibrium was observed for the bound ligand reflecting the dynamics of EF l...

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“…The low pK a value (1.05) estimated for Asp 98 in lacb_bovin is in good agreement with the value of 2 that was determined experimentally by NMR (20).…”

Section: Fig 2 Grid Docking Solutions Obtained For Blg (A) and Plg (B)supporting

confidence: 88%

mentioning

confidence: 99%

EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins

Ragona

Fogolari

Catalano

et al. 2003

Journal of Biological Chemistry

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“…Another interesting example of a perturbed logK value is that of the catalytic lysine in acetoacetate decarboxylase and polyamine enzyme models [224]. 1 H NMR titrations performed as 2D experiments to improve resolution lead to the determination of all (or nearly all) protonation constants of ribonuclease A [225], mouse epidermal growth factor [39], α-sarcin [226], and bovine β-lactoglobulin [227].…”

Section: Miscellaneousmentioning

confidence: 99%

Determination of microscopic acid?base parameters from NMR?pH titrations

Szakács

Kraszni

Noszál

2004

Analytical and Bioanalytical Chemistry

107

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The theory and practice of proton microspeciation based on NMR-pH titrations are surveyed. Principles of bi-, tri-, tetra-, and n-protic microequilibrium systems are discussed. Evaluation methods are exemplified by case studies on bi- and tetraprotic biomolecules. Selection criteria and properties of 'reporter' NMR nuclei are described. Literature data on complete microspeciations of small ligands and site-specific basicity characterizations of peptides and proteins are critically reviewed.

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“…Cysteine pK a Calculation-pK a values for cysteines were calculated, based on the Poisson-Boltzmann equation, essentially according to the method of Antosiewicz et al (58) with minor modifications (59). The starting structures were a model built by homology with Pax-6 (PDB code 6PAX) as template, and the 20 NMR structures.…”

Section: Methodsmentioning

confidence: 99%

The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family

Codutti

Ingen

Vascotto

et al. 2008

Journal of Biological Chemistry

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Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys 45 and Cys 57 ). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys 45 and Cys 57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors.Pax-8 is an important eukaryotic transcription factor that is responsible, during embryogenesis, for the differentiation of several organs such as kidney, thyroid, and neural tube. Pax-8 is also essential in the adult organism, where it activates thyroid hormone production. During development, Pax-8 functionality is regulated by alternative splicing, generating isoforms that exhibit different transactivation properties but maintain unaltered the DNA binding domain (1, 2). In mature thyroid follicular cells, the complete Pax-8 splicing isoform regulates the expression of thyroglobulin, thyroperoxidase (3, 4), sodium/ iodide symporter, and thyrotropin receptor genes (5-7). Pax-8 activity is co-regulated by thyroid transcription factor-1, thyroid transcription factor-2, and Hex, indicating an active role in the early commitment and differentiation of thyrocytes (8, 9). The Pax family shares a bipartite functionality consisting of a N-terminal binding region and a C-terminal transactivation region.The N-terminal region is usually comprised of three domains, namely a Paired Box (Prd) domain, a conserved octapeptide, and a further homeodomain (see Fig. 1). Differences in conservation and functionality in these domains have been used as evolution markers for the Pax family (10), which has been subdivided into four groups according to the sequence homology in the N-terminal region. In particular, the N-terminal region of Pax-8 is composed by all three domains but presents an incomplete and inactive homeodomain. The Prd domain consists of a well conserved 128-residue-long region, formed by two distinct subdomains known in literature as PAI (N-terminal) and RED (C-terminal) (11).DNA binding studies have demonstrated that the two subdomains of the Pax-8 Prd domain bind DNA independently (12), are both required f...

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Electrostatic properties of bovine ?-lactoglobulin

Cited by 79 publications

References 45 publications

EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins

EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins

Determination of microscopic acid?base parameters from NMR?pH titrations

The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family

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