NMR investigation of the equilibrium partitioning of a water-soluble bile salt protein carrier to phospholipid vesicles

Abstract

Membrane binding by cytosolic fatty acid binding proteins (FABP) appears to constitute a key step of intracellular lipid trafficking. We applied NMR spectroscopy to study the partitioning of a water-soluble bile acid binding protein (BABP), belonging to the FABP family, between its free and lipid-vesicle-bound states. As the lipid-bound protein was NMR-invisible, the signals of the free biomolecule were analyzed to obtain quantitative information on binding affinity and steady-state kinetics. The data indicated a reversible interaction of BABP with anionic vesicles occurring in a very slow exchange regime on the NMR time scale. The approximate binding epitope was demonstrated from results on BABP samples in which different positively charged lysine residues were mutated to neutral alanines. H/D exchange measurements indicated a higher exposure to solvent for the core amino acid residues in the liposome-bound state. Finally, the BABP-liposome interaction was also investigated for the first time through an MRI-chemical exchange saturation transfer experiment that has potential applications not only in the field of biology, but also in biomedicine, bioanalytical chemistry, and nanotechnology.

Structural and Molecular Determinants of Membrane Binding by the HIV-1 Matrix Protein

“…We employed a recently developed NMR approach [55,56] to monitor protein binding to liposomes. This approach allows measurement of the amount of protein that remains free in solution in the presence of liposomes under equilibrium conditions, and is based on the fact that NMR signals are readily detectable for proteins the size of HIV-1 MA (~15kDa) but generally undetectable (or “dark” [55]) for proteins and protein complexes as large as liposomes (>60MDa).…”

“…This approach allows measurement of the amount of protein that remains free in solution in the presence of liposomes under equilibrium conditions, and is based on the fact that NMR signals are readily detectable for proteins the size of HIV-1 MA (~15kDa) but generally undetectable (or “dark” [55]) for proteins and protein complexes as large as liposomes (>60MDa). Provided that on/off membrane exchange is intermediate-to-slow on the NMR chemical shift time scale, binding to liposomes is expected to render all but the highly flexible MA residues NMR-invisible [55,56]. As shown in Fig.…”

“…In the present studies, we probed for MA interactions with native PI(4,5)P 2 and other membrane constituents using a recently developed NMR-detected liposome binding assay [56]. In the absence of anionic phospholipids, MA exhibited low but detectable affinity for PC liposomes.…”

“…The fraction of protein bound was calculated as a function of the missing signal intensity for each lipid/protein molar ratio, discussed further [56]. The Gnuplot graphing utility (http://www.gnuplot.info) was used for graphing and non-linear least squares fitting of binding isotherms.…”

Are they in or out? The Elusive Interaction between Qtracker ® 800 Vascular Labels and Brain Endothelial Cells

“…DLS was performed on Qtracker ® 800 using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd, MA, USA) operating with a 633 nm He-Ne laser light source and a fixed detector angle of 173°, as previously described [27]. Qtracker ® 800, 2-μM stock solution of 2 μM in 50 mM borate buffer, pH 8.3 (Life Technologies) was diluted to the range of 40-80 nM either in Ringer solution or in saline solution (See section Chemicals).…”