دانلود رایگان مقاله اثر تراکم ماکرومولکولی بر پروتئین اتصالی میله ای کم چرب

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.

4. Conclusions

In this work, we provided a comprehensive phenomenological description of the impact of diverse crowding agents on the individual amino acid residues in a small dynamic lipid binding protein. Protein structure, local dynamics, and ligand binding mechanism were found to be minimally perturbed when direct proteincrowder interactions were absent. However, even the ‘inert’ polymer Ficoll appeared to influence the availability of long chain fatty acids for binding to LFABP. At variance with Ficoll, both PEG and LSZ associated specifically with the test protein. In agreement with previously described systems, BSA caused perturbations to relaxation rates, supporting the occurrence of weak unspecific interactions with LFABP. The test protein showed unique behaviour in complex environments, such as cell lysates and water-in-oil droplets, due to its tendency to interact with hydrophobic molecules and amphiphiles, also in competition with cognate ligands. The data suggest that promiscuous interactions may constitute a major factor affecting the function of lipid binding proteins in the cell.