Multiple Locations of Peptides in the Hydrocarbon Core of Gel-Phase Membranes Revealed by Peptide ¹³C to Lipid ²H Rotational-Echo Double-Resonance Solid-State Nuclear Magnetic Resonance

Membrane locations of peptides and proteins are often critical to their functions. Solid-state rotational-echo double-resonance (REDOR) nuclear magnetic resonance is applied to probe the locations of two peptides via peptide ¹³CO to lipid ²H distance measurements. The peptides are KALP, an α-helical membrane-spanning peptide, and HFP, the β-sheet N-terminal fusion peptide of the HIV gp41 fusion protein that plays an important role in HIV–host cell membrane fusion. Both peptides are shown to have at least two distinct locations within the hydrocarbon core of gel-phase membranes. The multiple locations are attributed to snorkeling of lysine side chains for KALP and to the distribution of antiparallel β-sheet registries for HFP. The relative population of each location is also quantitated. To the best of our knowledge, this is the first clear experimental support of multiple peptide locations within the membrane hydrocarbon core. These data are for gel-phase membranes, but the approach should work for liquid-ordered membranes containing cholesterol and may be applicable to liquid-disordered membranes with appropriate additional analysis to take into account protein and lipid motion. This paper also describes the methodological development of ¹³CO–²H REDOR using the lyophilized I4 peptide that is α-helical and ¹³CO-labeled at A9 and ²H_α-labeled at A8. The I4 spins are well-approximated as an ensemble of isolated ¹³CO–²H spin pairs each separated by 5.0 Å with a 37 Hz dipolar coupling. A pulse sequence with rectangular 100 kHz ²H π pulses results in rapid and extensive buildup of REDOR (Δ<i>S</i>/<i>S</i>₀) with a dephasing time (τ). The buildup is well-fit by a simple exponential function with a rate of 24 Hz and an extent close to 1. These parameter values reflect nonradiative transitions between the ²H spin states during the dephasing period. Each spin pair spends approximately two-thirds of its time in the ¹³CO–²H (<i>m</i> = ±1) states and approximately one-third of its time in the ¹³CO–²H (<i>m</i> = 0) state and contributes to the Δ<i>S</i>/<i>S</i>₀ buildup during the former but not the latter time segments

Copper-Catalyzed Oxidative Cross-Dehydrogenative Coupling/Oxidative Cycloaddition: Synthesis of 4‑Acyl-1,2,3-Triazoles

A copper-catalyzed three-component reaction of methyl ketones, organic azides, and various one-carbon (C1) donors was developed that provides 4-acyl-1,2,3-triazoles in moderate to good yields. While DMF, DMA, TMEDA, or DMSO can serve as the C1 donor, best yields were obtained using DMF. The transformation is proposed to proceed via an oxidative C–H/C–H cross-dehydrogenative coupling followed by an oxidative 1,3-dipolar cycloaddition

Closed and Semiclosed Interhelical Structures in Membrane vs Closed and Open Structures in Detergent for the Influenza Virus Hemagglutinin Fusion Peptide and Correlation of Hydrophobic Surface Area with Fusion Catalysis

The ∼25 N-terminal “HAfp” residues of the HA2 subunit of the influenza virus hemagglutinin protein are critical for fusion between the viral and endosomal membranes at low pH. Earlier studies of HAfp in detergent support (1) N-helix/turn/C-helix structure at pH 5 with open interhelical geometry and N-helix/turn/C-coil structure at pH 7; or (2) N-helix/turn/C-helix at both pHs with closed interhelical geometry. These different structures led to very different models of HAfp membrane location and different models of catalysis of membrane fusion by HAfp. In this study, the interhelical geometry of membrane-associated HAfp is probed by solid-state NMR. The data are well-fitted to a population mixture of closed and semiclosed structures. The two structures have similar interhelical geometries and are planar with hydrophobic and hydrophilic faces. The different structures of HAfp in detergent vs membrane could be due to the differences in interaction with the curved micelle vs flat membrane with better geometric matching between the closed and semiclosed structures and the membrane. The higher fusogenicity of longer sequences and low pH is correlated with hydrophobic surface area and consequent increased membrane perturbation

Copper-Catalyzed Cross-Dehydrogenative <i>N</i>²‑Coupling of <i>NH</i>-1,2,3-Triazoles with <i>N</i>,<i>N</i> -Dialkylamides: <i>N</i>‑Amidoalkylation of <i>NH</i>-1,2,3-Triazoles

An efficient copper-catalyzed C–N bond formation by N–H/C–H cross-dehydrogenative coupling (CDC) between <i>NH</i>-1,2,3-triazoles and <i>N</i>,<i>N</i>-dialkylamides has been developed. The method provided <i>N</i>-amidoalkylated 1,2,3-triazoles with moderate to high yields, and the reactions showed high <i>N</i>²-selectivities when 4,5-disubstituted <i>NH</i>-1,2,3-triazoles served as the substrates