Pro-arrhythmic effects of gain-of-function potassium channel mutations in the short QT syndrome.

The congenital short QT syndrome (SQTS) is a rare condition characterized by abbreviated rate-corrected QT (QTc) intervals on the electrocardiogram and by increased susceptibility to both atrial and ventricular arrhythmias and sudden death. Although mutations to multiple genes have been implicated in the SQTS, evidence of causality is particularly strong for the first three (SQT1-3) variants: these result from gain-of-function mutations in genes that encode K+ channel subunits responsible, respectively, for the IKr, IKs and IK1 cardiac potassium currents. This article reviews evidence for the impact of SQT1-3 missense potassium channel gene mutations on the electrophysiological properties of IKr, IKs and IK1 and of the links between these changes and arrhythmia susceptibility. Data from experimental and simulation studies and future directions for research in this field are considered. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

hERG1a/1b heteromeric currents exhibit amplified attenuation of inactivation in variant 1 short QT syndrome.

Potassium channels encoded by hERG (human ether-à-go-go-related gene) underlie the cardiac rapid delayed rectifier K+ current (IKr) and hERG mutations underpin clinically important repolarization disorders. Virtually all electrophysiological investigations of hERG mutations have studied exclusively the hERG1a isoform; however, recent evidence indicates that native IKr channels may be comprised of hERG1a together with the hERG1b variant, which has a shorter N-terminus. Here, for the first time, electrophysiological effects were studied of a gain-of-function hERG mutation (N588K; responsible for the 'SQT1' variant of the short QT syndrome) on current (I(hERG1a/1b)) carried by co-expressed hERG1a/1b channels. There were no significant effects of N588K on I(hERG1a/1b) activation or deactivation, but N588K I(hERG1a/1b) showed little inactivation up to highly positive voltages (< or = +80 mV), a more marked effect than seen for hERG1a expressed alone. I(hERG1a/1b) under action potential voltage-clamp, and the effects on this of the N588K mutation, also showed differences from those previously reported for hERG1a. The amplified attenuation of I(hERG) inactivation for the N588K mutation reported here indicates that the study of co-expressed hERG1a/1b channels should be considered when investigating clinically relevant hERG channel mutations, even if these reside outside of the N-terminus region.

Nanometer-scale ion aggregates in aqueous electrolyte solutions: guanidinium carbonate.

Neutron diffraction with isotopic substitution (NDIS) experiments and molecular dynamics (MD) simulations have been used to characterize the structure of aqueous guanidinium carbonate (Gdm2CO3) solutions. The MD simulations found very strong hetero-ion pairing in Gdm2CO3 solution and were used to determine the best structural experiment to demonstrate this ion pairing. The NDIS experiments confirm the most significant feature of the MD simulation, which is the existence of strong hetero-ion pairing between the Gdm+ and CO3(2-) ions. The neutron structural data also support the most interesting feature of the MD simulation, that the hetero-ion pairing is sufficiently strong as to lead to nanometer-scale aggregation of the ions. The presence of such clustering on the nanometer length scale was then confirmed using small-angle neutron scattering experiments. Taken together, the experiment and simulation suggest a molecular-level explanation for the contrasting denaturant properties of guanidinium salts in solution.

Nanometer-scale ion aggregates in aqueous electrolyte solutions: guanidinium sulfate and guanidinium thiocyanate.

Neutron diffraction experiments and molecular dynamics simulations are used to study the structure of aqueous solutions of two electrolytes: guanidinium sulfate (a mild protein conformation stabilizer) and guanidinium thiocyanate (a powerful denaturant). The MD simulations find the unexpected result that in the Gdm2SO4 solution the ions aggregated into mesoscopic (nanometer-scale) clusters, while no such aggregation is found in the GdmSCN solution. The neutron diffraction studies, the most direct experimental probe of solution structure, provide corroborating evidence that the predicted very strong ion pairing does occur in solutions of 1.5 m Gdm2SO4 but not in 3 m solutions of GdmSCN. A mechanism is proposed as to how this mesoscopic solution structure affects solution denaturant properties and suggests an explanation for the Hofmeister ordering of these solutions in terms of this ion pairing and the ability of sulfate to reverse the denaturant power of guanidinium.

The hydration structure of guanidinium and thiocyanate ions: implications for protein stability in aqueous solution.

Neutron diffraction experiments were carried out on aqueous solutions containing either guanidinium or thiocyanate ions. The first-order difference method of neutron diffraction and isotopic substitution was applied, and the hydration structures of two of nature's strongest denaturant ions were determined. Each ion is shown to interact weakly with water: Guanidinium has no recognizable hydration shell and is one of the most weakly hydrated cations yet characterized. Hydration of thiocyanate is characterized by a low coordination number involving around one hydrogen-bonded water molecule and approximately two water molecules weakly interacting through "hydration bonds." The weak hydration of these denaturant ions strongly supports suggestions that a major contribution to the denaturant effect is the preferential interaction of the denaturant with the protein surface. By contrast, solute species such as many sugars and related polyols that stabilize proteins are strongly hydrated and are thus preferentially retained in the bulk solvent and excluded from the protein surface.

Structure, location, and lipid perturbations of melittin at the membrane interface.

Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin's exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1.

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.

The asparagine-stabilized beta-turn of apamin: contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis.

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed.

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.

Intrinsic helical propensities and stable secondary structure in a membrane-bound fragment (S4) of the shaker potassium channel.

The location and stability of helical secondary structure in a fragment comprising an extended sequence of the S4 transmembrane segment of the Shaker potassium channel was determined in methanol, and when bound to vesicles composed of egg phosphatidylcholine: egg phosphatidylglycerol (4:1; mol:mol) in water. The N-acetylated, C-amidated peptide corresponds to the sequence comprising residues A355-I384 in the Shaker potassium channel. Although NOEs characteristic of helical structure encompass essentially the full peptide sequence in methanol, analysis of amide and CH(alpha) chemical shifts, and amide exchange protection factors establish that stable helical structure comprises only around the first 22 amino acids of the 30 residue peptide. This sequence corresponds to that predicted to have the highest helical stability in water, indicating that while helical structure is considerably stabilised in methanol, the relative helical propensities of amino acids in methanol may be similar to those in water. In the presence of vesicles containing negatively charged lipids, helical structure corresponding to a maximum of around 40 % of the extended S4 peptide is induced; no helical structure is induced in the presence of vesicles composed only of neutral lipids. The location of stable helical structure in the membrane-bound peptide was determined by amide hydrogen-deuterium exchange trapping, and was shown to encompass the sequence between residues near M2 and I18. This sequence is similar to that having high helix propensity in water and methanol, supporting the idea that intrinsic helical propensities are important in defining the location of stable helical structure in polypeptides bound in the interfacial region of lipid bilayers. The study defines an approach to determining the location of, and contributions to, the stability of helical secondary structure in membrane-reconstituted polypeptides.

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.

A general method for relieving substrate inhibition in lactate dehydrogenases.

The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure alpha-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD+ binary complex is not perturbed. (2) The mutation also leads to a large increase in the KM for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme-NADH complex.

Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide-dimerised melittin analogues.

The effects of covalent dimerisation of melittin by disulphide formation in cysteine-substitution analogues, (melittin K23C)2 and (melittin K23Q,Q25C)2, on the kinetics of pore formation in phosphatidylcholine small unilamellar vesicles was measured under low ionic strength conditions. The initial rate of melittin-induced pore formation increased with the square of the peptide concentration, whereas both disulphide-dimerised melittin analogues showed a first-order dependence of pore formation rates on peptide concentration. These results indicate that peptide dimerisation is rate-limiting for pore formation under these conditions. A model for a generalised bilayer perturbation resulting from the self-association of a pair of peptide helices at the membrane surface is proposed which may have implications for a number of biological processes that involve the interaction of helical polypeptides with membranes.

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol.

Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.

Acquisition of native beta-strand topology during the rapid collapse phase of protein folding.

The 98 residue C-terminal domain of the cell-surface receptor protein CD2 (CD2.D1) has a beta-sandwich fold belonging to the immunoglobulin superfamily but lacking the usual disulfide bridges. Kinetic studies on the folding/unfolding of CD2.D1 reveal that folding proceeds through a rapidly formed intermediate state [Parker, M. J., & Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. To characterize the structural properties of this intermediate we have performed a series of amide hydrogen exchange studies using the pH competition method, in which folding and exchange are initiated simultaneously. The complex beta-sheet topology of this molecule makes it an ideal object for examining the acquisition of backbone hydrogen bonds made between sequence-local and sequence-distant segments of the chain during folding. The pattern of protected amides in the intermediate reveal that the essential features of the beta-sheet topology of CD2.D1 are defined early in the folding pathway, before the development of intimate side chain interactions characteristic of the native state. The results are discussed in light of current issues concerning the mechanistic relevance of kinetic protein folding intermediates.

Helix bending in alamethicin: molecular dynamics simulations and amide hydrogen exchange in methanol.

Molecular dynamics simulations of alamethicin in methanol were carried out with either a regular alpha-helical conformation or the x-ray crystal structure as starting structures. The structures rapidly converged to a well-defined hydrogen-bonding pattern with mixed alpha-helical and 3(10)-helical hydrogen bonds, consistent with NMR structural characterization, and did not unfold throughout the 1-ns simulation, despite some sizable backbone fluctuations involving reversible breaking of helical hydrogen bonds. Bending of the helical structure around residues Aib10-Aib13 was associated with reversible flips of the peptide bonds involving G11 (Aib10-G11 or G11-L12 peptide bonds), yielding discrete structural states in which the Aib10 carbonyl or (rarely) the G11 carbonyl was oriented away from the peptide helix. These peptide bond reversals could be accommodated without greatly perturbing the adjacent helical structure, and intramolecular hydrogen bonding was generally maintained in bent states through the formation of new (non-alpha or 3[10]) hydrogen bonds with good geometries: G11 NH-V9 CO (inverse gamma turn), Aib13 NH-Aib8 CO (pi-helix) and, rarely, L12 NH- Q7 NH (pi-helix). These observations may reconcile potentially conflicting NMR structural information for alamethicin in methanol, in which evidence for conformational flexibility in the peptide sequence before P14 (G11-Aib13) contrasts with the stability of backbone amide NH groups to exchange with solvent. Similar reversible reorientation of the Thr11-Gly12 peptide bond of melittin is also observed in dynamics simulations in methanol (R. B. Sessions, N. Gibbs, and C. E. Dempsey, submitted). This phenomenon may have some role in the orientation of the peptide carbonyl in solvating the channel lumen in membrane ion channel states of these peptides.

Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2).

The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the polyketide synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first polyketide synthase component for which detailed structural information has been obtained. Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints. The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2. The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3. The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86). The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid synthase (FAS) ACP, though some structural differences exist. First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E. coli FAS ACP. Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E. coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35). Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing polyketide chain. The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.