Mid-Membrane Photolabeling of the Transmembrane Domain of Glycophorin A in Phospholipid Vesicles

Abstract

The topography of membrane-bound proteins at atomic resolution is known only in rare cases. [1] Although the primary amino-acid sequence of glycophorin A (GPA), the major sialoglycoprotein of the human erythrocytes, has been known for more than twenty years and was the first membrane protein sequence elucidated, [2] a three-dimensional picture of the protein is still missing. We have previously developed the photoactivable membrane probe 1. This is a phospholipid with two distal, polar heads (a bola-amphiphile) and carries a photosensitive group (benzophenone) in the middle of a transmembrane chain. It is easily incorporated into DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocoline) vesicles, where it spans the bilayer at least in the presence of physiological concentrations of cholesterol. We demonstrated recently that the tandem use of the probe 1 a and cholesterol (for its ordering effect) in photolabeling experiments on DMPC vesicles led to a remarkable regioselective functionalization and we are presently conducting such studies. With this information, we expect that the present strategy using Au/ Ag(upd) electrodes for halide detection will provide the notable abilities to measure the concentrations of multiple halides simultaneously and to provide specific signals for confirming their identification. Experimental SectionMaterials: Au (99.99 %) shot and Cr-coated tungsten filaments were obtained from Americana Precious Metals (East Rutherford, NJ) and R. D. Mathis (Long, Beach, CA), respectively. Sulfuric acid (double distilled, 98 %) and silver sulfate (c(Cl À ) 0.02 %) were obtained from Aldrich and used as received. KCl, KBr, and KI were obtained from Mallinckrodt and used as received. Au(111) samples were prepared by sequential evaporation of Cr (2 ± 3 nm) and Au (150 nm) onto glass slides and a post-evaporation annealing in a hydrogen flame. [12] Electrochemical measurements: Cyclic voltammetry was conducted with a computer-controlled PAR Model 263A potentiostat. A solution of 0.6 mm Ag 2 SO 4 and 0.1m H 2 SO 4 in deionized water (Millipore, 18.2 MW) was used to deposit the Ag upd adlayer on gold. Modified electrodes were prepared by cycling once in this solution and being removed at a 300 mV versus Ag / Ag 0 under potentiostatic control. [13] The Au(111)/Ag(upd) electrodes were rinsed with deionized water and immersed into a halide test solution for varying amounts of time. The samples were rinsed with water and characterized electrochemically in a 0.6 mm Ag 2 SO 4 /0.1m H 2 SO 4 (aq) solution. A standard three-electrode configuration was used to obtain all CVs, and all potentials are quoted relative to silver wire (Ag /Ag 0 ).

Solid-State NMR Investigation of the Depth of Insertion of Protegrin-1 in Lipid Bilayers Using Paramagnetic Mn2+

“…However, this technique requires the protein to have suitable mobility in the bilayer: mobile peptides have inefficient magnetization transfer rates from the lipids. Beyond NMR, electron paramagnetic resonance spectroscopy and optical spectroscopic methods such as fluorescence resonance energy transfer are well established for investigating the depth of insertion of membrane peptides (Altenbach et al, 1994;Lakey et al, 1993;Ogawa et al, 2001;Shin et al, 1993). However, these approaches require the use of bulky and often dynamic spin probes, which complicate the extraction of the immersion depth.…”

Photolabeling Strategies to Study Membranotropic Peptides Interacting with Lipids and Proteins in Membranes

“…They observed that most of the cross-linking involved the carboxyl group of Glu 70 , thus suggesting that this residue was located within the bilayer 23 . However, twenty years later the group of Nakatani used the same approach with a different lipid probe and obtained very different results 24,25 . They used a benzophenonebased bola-phospholipid probe (10, Figure 2), thus guaranteeing that the label was located at the center of bilayer, with no possibility for looping back.…”

Palladium(II)-catalyzed alkenyl C-H bond functionalization

“…FTIR (NaCl, cm -1 ): 3005, 2978, 2931, 2250, 1703, 1693, 1618, 1492, 1446, 1390, 1296, 1247, 1132 under normal conditions, the arylation or olefination products resulting from olefins with electron-rich substituents usually give rise to a mixture of both the α-and β-substituted regioisomeric olefins. 117 The regioselectivity of the Heck reaction has remained a hot research topic for many years.…”