Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

The T Cell Antigen Receptor α Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations.

Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.

Impact of Ho(3+)-doping on (13)C dynamic nuclear polarization using trityl OX063 free radical.

We have investigated the effects of Ho-DOTA doping on the dynamic nuclear polarization (DNP) of [1-(13)C] sodium acetate using trityl OX063 free radical at 3.35 T and 1.2 K. Our results indicate that addition of 2 mM Ho-DOTA on 3 M [1-(13)C] sodium acetate sample in 1 : 1 v/v glycerol : water with 15 mM trityl OX063 improves the DNP-enhanced (13)C solid-state nuclear polarization by a factor of around 2.7-fold. Similar to the Gd(3+) doping effect on (13)C DNP, the locations of the positive and negative (13)C maximum polarization peaks in the (13)C microwave DNP sweep are shifted towards each other with the addition of Ho-DOTA on the DNP sample. W-band electron spin resonance (ESR) studies have revealed that while the shape and linewidth of the trityl OX063 ESR spectrum was not affected by Ho(3+)-doping, the electron spin-lattice relaxation time T1 of trityl OX063 was prominently reduced at cryogenic temperatures. The reduction of trityl OX063 electron T1 by Ho-doping is linked to the (13)C DNP improvement in light of the thermodynamic picture of DNP. Moreover, the presence of Ho-DOTA in the dissolution liquid at room temperature has negligible reduction effect on liquid-state (13)C T1, in contrast to Gd(3+)-doping which drastically reduces the (13)C T1. The results here suggest that Ho(3+)-doping is advantageous over Gd(3+) in terms of preservation of hyperpolarized state-an important aspect to consider for in vitro and in vivo NMR or imaging (MRI) experiments where a considerable preparation time is needed to administer the hyperpolarized (13)C liquid.

Selective Membrane Disruption Mechanism of an Antibacterial γ-AApeptide Defined by EPR Spectroscopy.

γ-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. It is not clear how γ-AApeptides interact with bacterial membranes and alter lipid assembly, but such information is essential to understanding their antimicrobial activities and guiding future design of more potent and specific antimicrobial agents. Using electron paramagnetic resonance techniques, we characterized the membrane interaction and destabilizing mechanism of a lipo-cyclic-γ-AApeptide (AA1), which has broad-spectrum antibacterial activities. The analyses revealed that AA1 binding increases the membrane permeability of POPC/POPG liposomes, which mimic negatively charged bacterial membranes. AA1 binding also inhibits membrane fluidity and reduces solvent accessibility around the lipid headgroup region. Moreover, AA1 interacts strongly with POPC/POPG liposomes, inducing significant lipid lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon AA1 binding for liposomes mimicking mammalian cell membranes, which consist of neutral lipids and cholesterol. Our findings suggest that AA1 interacts and disrupts bacterial membranes through a carpet-like mechanism. The results showed that the intrinsic features of γ-AApeptides are important for their ability to disrupt bacterial membranes selectively, the implications of which extend to developing new antibacterial biomaterials.

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

High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(∼94 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.