title: Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level.
abstract: Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell.
authors.affiliation: Department of Biotechnology, Strada Le Grazie 15, University of Verona, 37134, Verona, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pérez Santero
authors.affiliation: Department of Biotechnology, Strada Le Grazie 15, University of Verona, 37134, Verona, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Favretto
authors.affiliation: Department of Biotechnology, Strada Le Grazie 15, University of Verona, 37134, Verona, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Zanzoni
authors.affiliation: Department of Biotechnology, Strada Le Grazie 15, University of Verona, 37134, Verona, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Chignola
authors.affiliation: Department of Biotechnology, Strada Le Grazie 15, University of Verona, 37134, Verona, Italy.
authors.forename: Michael
authors.initials: M
authors.lastname: Assfalg
authors.affiliation: Department of Biotechnology, Strada Le Grazie 15, University of Verona, 37134, Verona, Italy. Electronic address: mariapina.donofrio@univr.it.
authors.forename: Mariapina
authors.initials: M
authors.lastname: D'Onofrio
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004529:Egg White; D004926:Escherichia coli / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D057446:Hydrodynamics; D008024:Ligands; D008027:Light; D008055:Lipids / Q000737:chemistry*; D046911:Macromolecular Substances / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation; D012710:Serum Albumin, Bovine / Q000737:chemistry
chemical_list: D000596:Amino Acids; C493319:FABP1 protein, human; D050556:Fatty Acid-Binding Proteins; D008024:Ligands; D008055:Lipids; D046911:Macromolecular Substances; D011108:Polymers; D012710:Serum Albumin, Bovine; D009113:Muramidase
keywords: Lipid binding protein; Macromolecular crowding; NMR spectroscopy; Protein dynamics; Protein-ligand binding