Bis(monoacylglycero)phosphate Forms Stable Small Lamellar Vesicle Structures: Insights into Vesicular Body Formation in Endosomes

AbstractBis(monoacylglycero)phosphate (BMP) is an unusually shaped lipid found in relatively high percentage in the late endosome. Here, we report the characterization of the morphology and molecular organization of dioleoyl-BMP (DOBMP) with dynamic light scattering, transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and electron paramagnetic resonance spectroscopy. The morphology of hydrated DOBMP dispersions varies with pH and ionic strength, and DOBMP vesicles are significantly smaller in diameter than phosphatidylcholine dispersions. At neutral pH, DOBMP forms highly structured, clustered dispersions 500 nm in size. On the other hand, at acidic pH, spherically shaped vesicles are formed. NMR and spin-labeled electron paramagnetic resonance demonstrate that DOBMP forms a lamellar mesophase with acyl-chain packing similar to that of other unsaturated phospholipids. 31P NMR reveals an orientation of the phosphate group in DOBMP that differs significantly from that of other phospholipids. These macroscopic and microscopic structural characterizations suggest that the biosynthesis of BMP on the inner luminal membrane of maturing endosomes may possibly produce budded vesicles high in BMP content, which form small vesicular structures stabilized by the physical properties of the BMP lipid

Towards increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

This work was performed at the National High Magnetic Field Laboratory (NHMFL), which is supported by the National Science Foundation (DMR-1157490) and the State of Florida. L.S. acknowledges support from the National Institutes of Health (AI091693) and the NHMFL User Collaboration Grants Program (Award No. 5080). G.E.F. acknowledges support from the National Science Foundation (MCB-1329467) and the National Institutes of Health (GM105409 and S10RR031603). S.H. acknowledges support from the National Science Foundation (DMR-1309463). J.M.E acknowledges support from the National Science Foundation (DGE-0802270).High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W- (∼95 GHz) and D-band (∼140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to α-helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ∼50 μL, concentration sensitivities of 2-20 μM were achieved, representing a ∼10-fold enhancement compared to a cylindrical TE011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.PostprintPeer reviewe

Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA₃

IA3 is an intrinsically disordered protein (IDP) that becomes helical when bound to yeast proteinase A (YPRA) or in the presence of the secondary stabilizer 2,2,2-trifluoroethanol (TFE). Here, site-directed spin-labeling (SDSL) continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy and circular dichroism (CD) are used to characterize the TFE-induced helical conformation of IA3 for a series of spin-labeled cysteine scanning constructs and varied amino acid substitutions. Results demonstrate that the N-terminal concave helical surface of IA3, which is the buried interface when bound to YPRA, can be destabilized by the spin-label or bulky amino acid substitutions. In contrast, the helical tendency of IA3 is enhanced when spin-labels are incorporated into the convex, i.e., solvent exposed, surface of IA3. No impact of the spin-label within the C-terminal region was observed. This work further demonstrates the utility and sensitivity of SDSL CW-EPR for studies of IDPs. In general, care must be taken to ensure the spin-label does not interfere with native helical tendencies and these studies provide us with knowledge of where to incorporate spin-labels for future SDSL investigations of IA3

Natural Polymorphisms D60E and I62V Stabilize a Closed Conformation in HIV-1 Protease in the Absence of an Inhibitor or Substrate

HIV infection remains a global health issue plagued by drug resistance and virological failure. Natural polymorphisms (NPs) contained within several African and Brazilian protease (PR) variants have been shown to induce a conformational landscape of more closed conformations compared to the sequence of subtype B prevalent in North America and Western Europe. Here we demonstrate through experimental pulsed EPR distance measurements and molecular dynamic (MD) simulations that the two common NPs D60E and I62V found within subtypes F and H can induce a closed conformation when introduced into HIV-1PR subtype B. Specifically, D60E alters the conformation in subtype B through the formation of a salt bridge with residue K43 contained within the nexus between the flap and hinge region of the HIV-1 PR fold. On the other hand, I62V modulates the packing of the hydrophobic cluster of the cantilever and fulcrum, also resulting in a more closed conformation