Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand's perspective.

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4, and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states ('intermediately' and 'strongly' bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature but with remarkable differences between the three isoforms. Additionally, the more dynamic state ('intermediately bound') seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest to the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level.

Aqueous Ionic Liquid Mixtures as Minimal Models of Lipid Bilayer Membranes.

We introduce aqueous ionic liquid (IL) mixtures, specifically mixtures of 1-butyl-3-imidazoliumtetrafluoroborate (BMImBF4), with water as a minimal model of lipid bilayer membranes. Imidazolium-based ILs are known to form clustered nanoscale structures in which local inhomogeneities, micellar or lamellar structures, are formed to shield hydrophobic parts of the cation from the polar cosolvent (water). To investigate these nanostructures, dynamic light scattering (DLS) on samples with different mixing ratios of water and BMImBF4 was performed. At mixing ratios of 50% and 45% (v/v), small and homogeneous nanostructures can indeed be detected. To test whether, in particular, these stable nanostructures in aqueous mixtures may mimic the effects of phospholipid bilayer membranes, we further investigated their interaction with myelin basic protein (MBP), a peripheral, intrinsically disordered membrane protein of the myelin sheath. Using dynamic light scattering (DLS), continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and small-angle X-ray scattering (SAXS) on recombinantly produced, "healthy" charge variants rmC1WT and double cysteine variant C1S17CH85C, we find that the size and the shape of the determined nanostructures in an optimum mixture offer model membranes in which the protein exhibits native behavior. SAXS measurements illuminate the size and shape of the nanostructures and indicate IL-rich "beads" clipped together by functional MBP, one of the in vivo roles of the protein in the myelin sheath. All the gathered data combined indicate that the 50% and 45% aqueous IL mixtures can be described as offering minimal models of a lipid mono- or bilayer that allow native processing and potential study of at least peripheral membrane proteins like MBP.

Tuning Human Serum Albumin (HSA) Hydrogels through Albumin Glycation.

The changes of technological properties of albumin-based hydrogels induced by increasing degrees of post-translational modification of the protein are reported. Maillard-type modification of amino acids arginine and lysine of albumin is achieved through glyoxal as an α-dicarbonyl compound. The degrees of modification are fine-tuned using different molar ratios of glyoxal. Hydrogels are thermally induced by heating highly concentrated precursor solutions above the protein's denaturation temperature. While the post-translational modifications are determined and quantified with mass spectrometry, continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy shed light on the protein fatty acid binding capacity and changes thereof in solution and in the gel state. The viscoelastic behavior is characterized as a measure of the physical strength of the hydrogels. On the nanoscopic level, the modified albumins in low concentration solution reveal lower binding capacities with increasing degrees of modification. On the contrary, in the gel state, the binding capacity remains constant at all degrees of modifications. This indicates that the loss of fatty acid binding capacity for individual albumin molecules is partially compensated by new binding sites in the gel state, potentially formed by modified amino acids. Such, albumin glycation offers a fine-tuning method of technological and nanoscopic properties of these gels.

Hydrogels from serum albumin in a molten globule-like state.

We demonstrate that a molten globule-like (MG) state of a protein, usually described as a compact yet non-folded conformation that is only present in a narrow and delicate parameter range, is preserved in the high concentration environment of the protein hydrogel. We reveal mainly by means of electron paramagnetic resonance (EPR) spectroscopy that bovine serum albumin (BSA) retains the known basic MG state after a hydrogel has been formed from 20 wt% precursor solutions. At pH values of ~11.4, BSA hydrogels made from MG-BSA remain stable for weeks, while gels formed at slightly different (~0.2) pH units above and below the MG-state value dissolve into viscous solutions. On the contrary, when hydrophobic screening agents are added such as amphiphilic, EPR-active stearic acid derivatives (16-DOXYL-stearic acid, 16-DSA), the MG-state based hydrogel is the least long-lived, as the hydrophobic interaction of delicately exposed hydrophobic patches of BSA molecules is screened by the amphiphilic molecules. These bio- and polymer-physically unexpected findings may lead to new bio-compatible MG-based hydrogels that display novel properties in comparison to conventional gels.