Mechanistic insight into human ether-à-go-go-related gene (hERG) K+ channel deactivation gating from the solution structure of the EAG domain

Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.     

Stacked Sets of Parallel, In-register ��-Strands of ��2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β₂-microglobulin (β₂m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β₂m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β₂m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β₂m consist of about six β₂m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.     

Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.     

Application of the osmotic virial equation in cryobiology

The multisolute osmotic virial equation is the only multisolute thermodynamic solution theory that has been derived from first principles and can make predictions of multisolute solution behaviour in the absence of multisolute solution data. Other solution theories either (i) include simplifying assumptions that do not take into account the interactions between different types of solute molecules or (ii) require fitting to multisolute data to obtain empirical parameters. The osmotic virial coefficients, which are obtained from single-solute data, can be used to make predictions of multisolute solution osmolality. The osmotic virial coefficients for a range of solutes of interest in cryobiology are provided in this paper, for use with concentration units of both molality and mole fraction, along with an explanation of the background and theory necessary to implement the multisolute osmotic virial equation.     

Effect of anionic polysaccharides on conformational changes and antioxidant properties of protein-polyphenol binary covalently-linked complexes

This work evaluated the anionic polysaccharides to improve the functional properties and antioxidant activities, and compared the effects on covalently-linked the soy protein isolate (SPI)- (–)-epigallocatechin-3-gallat (EGCG) binary complexes. The increasing molecular weight via covalent insertion of (–)-epigallocatechin-3-gallat (EGCG) into the soy protein isolate (SPI) was verified by SDS-PAGE. The addition of polysaccharides had no obvious effects on the molecular weight, indicating noncovalent insertion by adsorption. Fourier transform infrared spectroscopy (FT-IR) analysis suggested that SPI-EGCG complexes mixed with polysaccharides changed the secondary structures of SPI with a decrease in α-helix and an increase in β-turn. The emulsions exhibited better stability index (ESI) by adding polysaccharides than the binary complexes emulsion, with decreased particle sizes and increased absolute ζ-potential values, while the value of ESI for the pectin mixed increased more. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging capacity of SPI-EGCG complexes with the addition of polysaccharides were both greater than 70 %. These findings indicated that anionic polysaccharides could be potentially used as a natural and safe alternative for regulating covalently-linked SPI-EGCG emulsion stability and improving emulsion oxidation resistance.     

Conformation of antibiotic X-537A (lasalocid-A) and its calcium complex in acetonitrile solvent using circular dichroism spectroscopy

The calcium binding characteristics of antibiotic X-537A (lasalocid-A) in a lipophilic solvent, acetonitrile (CH3CN), have been studied using circular dichroism (CD) spectroscopy. The analysis of the data indicated that in this medium polar solvent, X-537A forms predominantly the charged complexes of stoichiometries 2:1 and 1:1, the relative amounts of the two being dependent on [Ca2+]. The conformations of the complexes, arrived at on the basis of the data, seem to indicate a rigid part encompassing Ca2+, liganded to 3 oxygens of the molecule, viz., the carbonyl, the substituted tetrahydrofuran ring and the substituted pyran ring oxygens (apart from, possibly, the liganding provided by nitrogen atoms of the solvent molecules), and a flexible part consisting of the salicylic acid group of the molecule.     

A Nucleotide-dependent and HRDC Domain-dependent Structural Transition in DNA-bound RecQ Helicase

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF₄. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.     

Heterocycles: Versatile control elements in bioactive macrocycles

The potential of macrocyclic peptides as therapeutics has garnered much attention over the last several years. Unlike their linear counterparts, macrocycles have higher resistance to enzymatic degradation and often display improved bioavailability. However, macrocycles are typically not lipophilic enough for cellular membrane penetration, which prevents them from interacting with intracellular targets. Methods to increase cellular permeability have involved the incorporation of bicyclic scaffolds, d-amino acids and N-methylation of amides. These modifications exert their effect through conformational control of macrocycles and have been well studied in the literature. In contrast, the structural consequences of heterocycle incorporation into macrocyclic rings has not been as exhaustively investigated. In this mini-review we discuss key examples in which heterocycles influence the conformational stability and other properties of macrocycles.     

Molecular dynamics simulations of unsaturated lipid bilayers: effects of varying the numbers of double bonds

The importance of unsaturated, and especially polyunsaturated phosphatidylcholine molecules for the functional properties of biological membranes is widely accepted. Here, the effects of unsaturation on the nanosecond-scale structural and dynamic properties of the phosphatidylcholine bilayer were elucidated by performance of multinanosecond molecular dynamics simulations of all-atom bilayer models. Bilayers of dipalmitoylphosphatidylcholine and its mono-, di-, and tetraunsaturated counterparts were simulated, containing, respectively, oleoyl, linoleoyl, or arachidonoyl chains in the sn-2 position. Analysis of the simulations focused on comparison of the structural properties, especially the ordering of the chains in the membranes. Although the results suggest some problems in the CHARMM force field of the lipids when applied in a constant pressure ensemble, the features appearing in the ordering of the unsaturated chains are consistent with the behaviour known from ²H NMR experiments. The rigidity of the double bonds is compensated by the flexibility of skew state single bonds juxtaposed with double bonds. The presence of double bonds in the sn-2 chains considerably reduces the order parameters of the CH bonds. Moreover, the double bond region of tetraunsaturated chains is shown to span all the way from the bilayer centre to the head group region.     

基于HP模型的蛋白质折叠问题的研究

基于蛋白质二维HP模型提出改进的遗传算法对真实蛋白质进行计算机折叠模拟。结果显示疏水能量函数最小值的蛋白质构象对应含疏水核心的稳定结构,疏水作用在蛋白质折叠中起主要作用。研究表明二维HP模型在蛋白质折叠研究中是可行的和有效的并为进一步揭示蛋白质折叠机理提供重要参考信息。