Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin

Bonarek, Piotr; Loch, J.I.; Tworzydło, Magdalena; Cooper, David R.; Milto, Katažyna; Wróbel, Paulina; Kurpiewska, Katarzyna; Lewiński, K.

doi:10.1016/j.jsb.2020.107493

Cited by 13 publications

(43 citation statements)

References 78 publications

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“…In both variants, the binding site was shorter than in the wild-type (WT) protein. The presence of a rigid aromatic ring introduced at position 56 prevented the binding of fatty acids by I56F variant, while in F105L variant the binding ability was retained due to the higher flexibility of the binding pocket (Bonarek et al, 2020). Interestingly, fatty acid bound by F105L variant had unusual bent conformation, not observed previously in any other lactoglobulin structures, indicating adjustment of both binding pocket and ligand conformation.…”

Section: Introductionmentioning

confidence: 75%

“…Variants L39Y, L58F and M107L retained the elongated shape of the binding pocket, very similar to the one present in WT protein. Their ability to bind fatty acids was confirmed by crystal structures revealing the presence of a single fatty acid molecule bound in an extended conformation in the β-barrel (Bonarek et al, 2020).…”

Section: Introductionmentioning

confidence: 92%

“…Human lipocalins are usually modified by randomization of flexible loops surrounding β-barrel to create highly specific proteins with potential medical applications called Anticalins (Rothe & Skerra, 2018). In our studies, local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change its ligand preferences (Loch et al, 2018;Bonarek et al, 2020). Detailed physicochemical characteristics of seven new lactoglobulin mutants (L39Y, I56F, L58F, V92F, V92Y, F105L, and M107L) as well as crystal structure of five of these variants and their complexes with selected fatty acids have been published elsewhere (Bonarek et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…In our studies, local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change its ligand preferences (Loch et al, 2018;Bonarek et al, 2020). Detailed physicochemical characteristics of seven new lactoglobulin mutants (L39Y, I56F, L58F, V92F, V92Y, F105L, and M107L) as well as crystal structure of five of these variants and their complexes with selected fatty acids have been published elsewhere (Bonarek et al, 2020). Crystallographic investigations revealed significant changes of the binding pocket shape in two mutants, I56F and F105L.…”

Section: Introductionmentioning

confidence: 99%

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Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket

et al. 2021

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β-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the β-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.

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Section: Introductionmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket

et al. 2021

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“…β-Lactoglobulin naturally occurs as a non-covalently bound dimer and this dimerization was found to play a critical role determining the affinity of β-lactoglobulin to ligands [ 14 ]. Recently, the natural affinity of β-lactoglobulin for fatty acids and other hydrophobic ligands initiated its exploitation to facilitate its potential as a drug carrier [ 15 , 16 , 17 ]. Other suggested biological roles of β-lactoglobulin include neonatal passive immunity transfer or a role in metabolism of phosphate in the mammary gland as a result of its observed interaction with p-nitrophenyl phosphate [ 18 ].…”

Section: β-Lactoglobulin Secretion Structure and Genetic Variantmentioning

confidence: 99%

Milk Processing Affects Structure, Bioavailability and Immunogenicity of β-lactoglobulin

Broersen

2020

Foods

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Bovine milk is subjected to various processing steps to warrant constant quality and consumer safety. One of these steps is pasteurization, which involves the exposure of liquid milk to a high temperature for a limited amount of time. While such heating effectively ameliorates consumer safety concerns mediated by pathogenic bacteria, these conditions also have an impact on one of the main nutritional whey constituents of milk, the protein β-lactoglobulin. As a function of heating, β-lactoglobulin was shown to become increasingly prone to denaturation, aggregation, and lactose conjugation. This review discusses the implications of such heat-induced modifications on digestion and adsorption in the gastro-intestinal tract, and the responses these conformations elicit from the gastro-intestinal immune system.

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β‐Lactoglobulin variants as potential carriers of pramoxine: Comprehensive structural and biophysical studies

Bonarek,

Mularczyk,

Loch

et al. 2023

J of Molecular Recognition

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Abstractβ‐Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to β‐lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X‐ray crystallography were used to understand the mechanisms of BLG–PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80‐fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the β‐barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.

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Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin

Cited by 13 publications

References 78 publications

Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket

Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket

Milk Processing Affects Structure, Bioavailability and Immunogenicity of β-lactoglobulin

β‐Lactoglobulin variants as potential carriers of pramoxine: Comprehensive structural and biophysical studies

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