The RCK2 domain of the human BKCa channel is a calcium sensor

Large conductance voltage and Ca2+-dependent K+ channels (BKCa) are activated by both membrane depolarization and intracellular Ca2+. Recent studies on bacterial channels have proposed that a Ca2+-induced conformational change within specialized regulators of K+ conductance (RCK) domains is responsible for channel gating. Each pore-forming α subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca2+ sensitivity, possibly participating in the formation of a Ca2+-binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca2+-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BKCa and prokaryotic RCK domains that reveal the location of a second RCK domain in human BKCa channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca2+-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca2+-induced change in conformation, associated with an α-to-β structural transition. We also show that the Ca bowl is an essential element for the Ca2+-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca2+-dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed

The Kinetics of the Hydrogen/Deuterium Exchange of Epidermal Growth Factor Receptor Ligands

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 310-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-α (TGF-α), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min−1 (Tyr), 0.09 min−1 (Arg, β-turns), and 1.88 × 10−3 min−1 (β-sheets and 310-helix); and for TGF-α 0.91 min−1 (Tyr), 0.27 min−1 (Arg, β-turns), and 1.41 × 10−4 min−1 (β-sheets). The time constants for AR 0.47 min−1 (Tyr), 0.04 min−1 (Arg), and 1.00 x 10−4 min−1 (buried 310-helix, β-turns, and β-sheets); for HB-EGF 0.89 min−1 (Tyr), 0.14 min−1 (Arg and 310-helix), and 1.00 x 10−3 min−1 (buried 310-helix, β-sheets, and β-turns); and for epiregulin 0.16 min−1 (Tyr), 0.03 min−1 (Arg), and 1.00 x 10−4 min−1 (310-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Biochimica et Biophysica Acta - Biomembranes 1788 (2009): 1255-1262, doi:10.1016/j.bbamem.2008.12.016.Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na+/Ca2+ exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far- UV circular dichroism and infrared spectra suggest a majority of β-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten β-sheets and two short α- helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.The work was supported by Grants from the US National Science Foundation [MCB 0444598], Fondo Nacional para Investigaciones Científicas y Tecnológicas [PICT-05- 12397 and PICT-05-38073], Consejo Nacional de Investigfaciones Científicas y Técnicas [PIP 5118 and PIP 5593] Secretaría de Ciencia y Técnica Universidad Nacional de Córdoba, Argentina, Fondo Nacional para Ciencia y Técnica [S1-9900009046 and G- 2001000637] and Fundación Polar, Venezuela and The Rhode Island Idea Network of Biomedical Research Excellence (INBRE)

More sustainable electricity generation in hot and dry fuel cells with a novel hybrid membrane of Nafion/nano-silica/hydroxyl ionic liquid

A new hybrid proton exchange membrane (PEM) has been prepared from hydroxyl functionalized imidazolium ionic liquid (IL-OH), Nafion and nano-SiO2. The IL-OH, with a hydroxyl group that acts as both a proton acceptor and donor, forms strong hydrogen bonds with both Nafion and nano-SiO2, resulting in an effective hydrogen bond network in the ternary membrane. Such an anhydrous hydrogen-bond network, which is unknown previously, endows the PEMs with higher proton conductivity, greater thermal stability and surprisingly a more robust mechanical performance than PEMs consisting of conventional ionic liquids. The resulting PEMs have a tensile strength that is more than twice as strong as recast Nafion and an anhydrous ionic conductivity of ∼55 mS cm−1 at temperatures above 160 °C, with a proton transfer number of ∼0.9. A laboratory assembled H2–O2 fuel cell employing this new PEM delivered a power density of 340 and 420 mW cm−2 at 160 and 180 °C, respectively

Measuring proteins in H2O with 2D-IR spectroscopy

The amide I infrared band of proteins is highly sensitive to secondary structure, but studies under physiological conditions are prevented by strong, overlapping water absorptions, motivating the widespread use of deuterated solutions. H/D exchange raises fundamental questions regarding the impact of increased mass on protein dynamics, while deuteration is impractical for biomedical or commercial applications of protein IR spectroscopy. We show that 2D-IR spectroscopy can avoid this problem because the 2D-IR amide I signature of proteins dominates that of water even at sub-millimolar protein concentrations. Using equine blood serum as a test system, we investigate the significant implications of being able to measure the spectroscopy and dynamics of proteins in water, demonstrating relevance in areas ranging from fundamental science to the clinic. Measurements of vibrational relaxation dynamics of serum proteins reveals that deuteration slows down the rate of amide I vibrational relaxation by >10%, indicating a dynamic impact of isotopic exchange in some proteins. The unique link between protein secondary structure and 2D-IR amide I lineshape allows differentiation of signals due to albumin and globulin protein fractions in serum leading to measurements of the biomedically-important albumin to globulin ratio (AGR) with an accuracy of ±4% across a clinically-relevant range. Furthermore, we demonstrate that 2D-IR spectroscopy enables differentiation of the structurally similar globulin proteins IgG, IgA and IgM, opening up a straightforward spectroscopic approach to measuring levels of serum proteins that are currently only accessible via biomedical laboratory testing

Thermo-oxidative degradation of additive free polyethylene. Part I. Analysis of chemical modifications at molecular and macromolecular scales

The effects of thermal oxidation on the molecular and macromolecular structures of additive free PE were investigated between 100°C and 140°C in air in order to tentatively establish non-empirical structure/property relationships. In the first part, the changes in POOH concentration were assessed by three different analytical methods: iodometry, modulated differential scanning calorimetry, and Fourier transform infrared (FTIR) spectrophotometry after SO2 treatment. All these methods provided very similar results until the end of the induction period, after which iodometry overestimated strongly POOH concentration because it titrates also other chemical species formed at high conversion ratios, namely double bonds. In parallel, the changes in carbonyl group concentration were determined by FTIR spectrophotometry after NH3 treatment. As the accumulation kinetics of ketones, aldehydes, and carboxylic acids were closely interrelated, the question of their actual formation mechanisms in the current thermal oxidation mechanistic scheme was raised. An alternative reaction pathway was proposed for the bimolecular decomposition of POOH. In the second part, the corresponding changes in weight and number average molecular masses were monitored by high temperature gel permeation chromatography equipped with a triple detection technology. As both quantities decreased dramatically from the beginning of exposure and their ratio Mw/Mn tends toward the asymptotic value of 2 and it was concluded that a "pure" chain scission process operated. Finally, as the number of chain scissions perfectly correlates, the concentration sum of aldehydes and their oxidation products (i.e., carboxylic acids), it was also concluded that these carbonyl groups result exclusively from the β scission of alkoxy radicals