KCNE1 (minK), found in the human heart and, cochlea is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of POPC and POPG was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (KV7.1) voltage-gated potassium channel led to native-like modulation of the channel. CD spectroscopy demonstrated that the %-helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles relative to the previously described structure in 1.0% LMPG micelles. SDSL EPR spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu 59, and Ser64 KCNE1 in both POPC/POPG vesicles and in LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu 59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water-inaccessible in vesicles, but found to be water-accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers, but also demonstrates the need to refine this structure using data derived from bilayer-reconstituted protein in order to more accurately define its native structure. This work establishes the basis for such future studies.

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the a-helical M2d subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine-mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i 1 1 to i 1 4). The characteristic periodicity of the a-helical structure gives rise to a unique pattern in the ESEEM spectra. In the i 1 1 and i 1 2 samples, the 2

Phospholamban (PLB) is an integral membrane protein regulating Ca2+ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca2+ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with 2H and 15N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). 2H and 15N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD3-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca2+-ATPase; thus, showing increased inhibition of Ca2+-ATPase.

Highlights d Synthetic antibodies that bivalently bind to HIV-1 can markedly enhance avidity d Linkers that enable bivalent binding are fully characterized by the linker entropy d Properly sized rigid linkers outperform long, flexible linkers d Avidity can be further enhanced by increasing antibody valency

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We have obtained residue-specific environmental parameters in the presence of the antiviral drug amantadine to gain information about the drug bound state of M2 in POPC/ POPG lipid bilayers. Power saturation studies of spin-labeled peptides reconstituted in a DOGS-NTA(Ni)-containing bilayers report on the accessibility of spin labels to nickel(II) chelated at the aqueous-lipid interface.