Denatured states of low-complexity polypeptide sequences differ dramatically from those of foldable sequences

How the primary sequence of a protein encodes conformational preferences that operate early in folding to promote efficient formation of the correct native topology is still poorly understood. To address this issue, we have prepared a set of yeast iso-1-cytochrome c variants that contain polyalanine inserts ranging from 6 to 30 residues in length near the N terminus of the protein. We study the thermodynamics and kinetics of His-heme loop formation in the denatured state at 3 and 6 M guanidine-HCl concentration. We find that polyalanine closely approximates a random coil with excluded volume giving scaling exponents, ν₃, for equilibrium loop formation of 2.26 ± 0.13 and 1.97 ± 0.04 in 3 and 6 M guanidine-HCl, respectively. The rate of loop breakage initially decreases and then becomes independent of loop size as would be expected for a random coil. Comparison with previously reported data for denatured state His-heme loop formation for iso-1-cytochrome c and Rhodopseudomonas palustris cytochrome c′, shows that foldable sequences deviate significantly from random coil behavior and that the deviation is fold-dependent.     

Transiently populated intermediate functions as a branching point of the FF domain folding pathway

Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module—the FF domain—with an equilibrium population of 2–3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K_d = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1·10⁴ M^-1 s^-1 and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.     

An attempt to distinguish between the actions of neuromuscular blocking drugs on the acetylcholine receptor and on its associated ionic channel

The effects of lobeline and tubocurarine on the voltage-clamped endplates of frog sartorius and cutaneous pectoris muscles were examined at room temperature (20-23°C). Like tubocurarine, lobeline causes nondepolarizing neuromuscular blockade. The half-time of decay (t_½) of endplate currents (e.p.c.s) recorded at a holding potential (V_m) of -90 mV was significantly shorter in endplates treated with lobeline (50 μM; mean t_½ ± SEM = 0.41 ± 0.02 ms) or tubocurarine (11.4 μM; t_½ = 0.64 ± 0.04 ms) than in those treated with Mg²⁺ (13 mM; t_½ = 1.39 ± 0.11 ms) or a low concentration of tubocurarine (3 μM; t_½ = 0.87 ± 0.05 ms). Similarly, lobeline (10 μM) shortened the t_½ of untreated miniature e.p.c.s by 35%; tubocurarine, however, abolished miniature e.p.c.s at the concentration required to observe its actions on e.p.c. decay kinetics. The t_½ of e.p.c.s recorded from preparations treated with Mg²⁺ (13 mM), tubocurarine at low concentrations (3 μM), or untreated miniature e.p.c.s was logarithmically related to V_m, being slower at more hyperpolarized values. By contrast, the t_½s of e.p.c.s recorded in either lobeline (50 μM) or tubocurarine (11.4 μM) were independent of voltage in the range -150 to -80 mV. The ability of lobeline to shorten t_½ and to remove the voltage dependence of t_½ was partially antagonized by Mg²⁺ (13 mM). As expected, when lobeline or tubocurarine was removed from the bath or when acetylcholine release from the motor nerve terminals was increased by 4-aminopyridine (20 μM) and Ca²⁺ (10 mM) (in the presence of lobeline or tubocurarine), the amplitude of e.p.c.s increased as a function of time. However, the t_½ of the decay phase of the e.p.c.s remained shortened (i.e., unaltered from the earlier treatment). These results suggest that both tubocurarine and lobeline have at least two distinct postjunctional actions including: (i) a block of the acetylcholine receptor and (ii) a block of the ionic channel associated with the acetylcholine receptor.     

From the Cover: Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high–free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50–90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.Determination of the mechanism of protein folding [unfolded (U) → folded (F)] is a long-standing goal of biophysical research. Folding of a single domain globular protein is a very highly cooperative (thermodynamically two-state) process. From analysis of folding kinetic data for CI2 and barnase, Fersht et al. (1) concluded that proteins fold through a high–free-energy transition state (TS) with partially formed elements of native structure. Recently, Barrick and Sosnick (2) concluded that an ensemble of unstable, rapidly reversible intermediates form early in the folding mechanism, and that the most advanced and unstable of these undergoes a rate-determining conformational change with transition state TS. This transit step, slower than the reverse direction of previous rapidly reversible steps, is followed by rapid propagation of folding. Most proposals for the initial intermediates invoke unstable regions of α-helix and/or β-sheet; these are thought to coalesce and/or rearrange to form TS. Kay and coworkers (3) characterized a marginally stable early intermediate of Fyn SH3 domain and found that interactions of amide groups formed earlier in the folding pathway than interactions of methyl groups, indicating that 2° structure formed before the 3° fold. Recent hydrogen exchange pulse-labeling experiments analyzed with mass spectrometry by Englander, Marqusee, and collaborators (4) indicate that folding of RNase H occurs through a defined set of intermediates where each step of folding adds more native-like elements of 2° structure. Late intermediates in folding that contain most elements of native structure except side-chain close packing have been observed (5).Mutational analyses of many proteins indicate that TS for protein folding are quite advanced (2). Here, we develop a different approach to characterize TS for noncovalent self-assembly processes like protein folding using solute effects and heat capacity changes as physical probes yielding amounts of hydrocarbon and amide surface buried in folding to and from TS, and apply it to characterize TS and infer properties of intermediates before TS for folding of 13 proteins. In addition to probing the character of TS in the protein-folding mechanism in a unique way, this research serves as a demonstration of the use of solutes as probes of conformational changes and interface formation in the steps of any protein process.The vast majority of the accessible surface area (ASA) buried in folding an extended polypeptide into a globular protein is either hydrocarbon (H) (∼70%) or amide (A) (∼20%), with an average ratio ΔASA_H/ΔASA_A ∼ 3.5 for complete folding (6). [Structural values of ΔASA_H/ΔASA_A range from 2.7 to 3.9 for 12 of the proteins analyzed here; only one (FKBP) has a ratio outside this range (∼5).] Different folding mechanisms lead to very different predictions of the ΔASA_H/ΔASA_A ratio for folding from an extended chain to TS. Formation of isolated elements of 2° structure (particularly α-helix) buries proportionately less hydrocarbon surface and proportionately more amide surface than does overall folding. For example, ΔASA_H/ΔASA_A is 0.6 for formation of an (AEAAKA)_n α-helix (6) and 2.0 for formation of isolated α-helical elements present in phage 434 cro repressor (calculation described below), whereas for β-hairpin [Protein Data Bank (PDB) ID code 2EVQ] formation ΔASA_H/ΔASA_A ∼ 2.7. For hydrophobic collapse mechanisms, the ratio ΔASA_H/ΔASA_A for folding to TS is presumably as large or larger than the value for complete folding.     

Extremely slow intramolecular diffusion in unfolded protein L

A crucial parameter in many theories of protein folding is the rate of diffusion over the energy landscape. Using a microfluidic mixer we have observed the rate of intramolecular diffusion within the unfolded B1 domain of protein L before it folds. The diffusion-limited rate of intramolecular contact is about 20 times slower than the rate in 6 M GdnHCl, and because in these conditions the protein is also more compact, the intramolecular diffusion coefficient decreases 100–500 times. The dramatic slowdown in diffusion occurs within the 250 μs mixing time of the mixer, and there appears to be no further evolution of this rate before reaching the transition state of folding. We show that observed folding rates are well predicted by a Kramers model with a denaturant-dependent diffusion coefficient and speculate that this diffusion coefficient is a significant contribution to the observed rate of folding.     

Fast events in protein folding: Relaxation dynamics of secondary and tertiary structure in native apomyoglobin

We report the fast relaxation dynamics of “native” apomyoglobin (pH 5.3) following a 10-ns, laser-induced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales (ν = 1633 cm⁻¹, τ = 48 ns; ν = 1650 cm⁻¹, τ = 132 μs). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm⁻¹) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm⁻¹). Folding rate coefficients inferred from the observed relaxations at 60°C are k_f(solvated) = (7 to 20) × 10⁶ s⁻¹ and k_f(native) = 3.6 × 10³ s⁻¹, respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60°C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.     

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (ΔH⁰ = 35 ± 4 kJ/mol) which is nearly compensated in free energy by the entropy change (ΔS⁰ = 112 ± 20 J·mol^-1·K^-1). The unlocking reaction has a time constant of about 1 μs at 5 °C and is enthalpy-limited with an activation energy of 32 ± 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 × 10¹¹ s^-1. In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 ± 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.     

Dependence of Heat Transport in Solids on Length-Scale,Pressure, and Temperature: Implications for Mechanisms and Thermodynamics

Accurate laser-flash measurements of thermal diffusivity (D) of diverse bulk solids at moderate temperature (T), with thickness L of ~0.03 to 10 mm, reveal that D(T) = D_∞(T)[1 − exp(−bL)]. When L is several mm, D_∞(T) = FT^−G + HT, where F is constant, G is ~1 or 0, and H (for insulators) is ~0.001. The attenuation parameter b = 6.19D_∞^−0.477 at 298 K for electrical insulators, elements, and alloys. Dimensional analysis confirms that D → 0 as L → 0, which is consistent with heat diffusion, requiring a medium. Thermal conductivity (κ) behaves similarly, being proportional to D. Attenuation describing heat conduction signifies that light is the diffusing entity in solids. A radiative transfer model with 1 free parameter that represents a simplified absorption coefficient describes the complex form for κ(T) of solids, including its strong peak at cryogenic temperatures. Three parameters describe κ with a secondary peak and/or a high-T increase. The strong length dependence and experimental difficulties in diamond anvil studies have yielded problematic transport properties. Reliable low-pressure data on diverse thick samples reveal a new thermodynamic formula for specific heat (∂ln(c_P)/∂P = −linear compressibility), which leads to ∂ln(κ)/∂P = linear compressibility + ∂lnα/∂P, where α is thermal expansivity. These formulae support that heat conduction in solids equals diffusion of light down the thermal gradient, since changing P alters the space occupied by matter, but not by light.     

SOD1 aggregation in ALS mice shows simplistic test tube behavior

A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.So far, the difficulty to experimentally measure protein aggregation in live tissue has focused many researchers to infer mechanistic details of neurodegenerative disease from molecular studies in vitro. An important outcome of this in vitro development is the establishment of rational protocols for quantitative assessment of protein aggregation data (1–4), which now start to consolidate our view of what is happening (5). Protein aggregation follows general and simplistic rules dictated by the amino acid sequence. However, the sheer number of competing aggregation sites within a typical protein chain (6) makes the process intrinsically malleable and dependent on experimental conditions (7). The nagging concern is then to what extent these already complex in vitro data are transferable to the even more complex situation in vivo? Here, we shed light on this question by comparing directly in vitro aggregation kinetics with corresponding data from transgenic mice using a recently developed in vivo quantification strategy based on antibodies (8). Our model system is the aggregation of superoxide dismutase 1 (SOD1) associated with the motor neuron disease ALS (8) (Fig. 1). A key feature of this system is that the immature apoSOD1 monomer, which is also implicated as a precursor in human pathology (9–12), needs to be globally unfolded to fibrillate in vitro (7) (Fig. 1). This simplistic behavior presents the experimental advantage that the fibrillation kinetics of apoSOD1 show simple dependence on structural stability (13, 14):ΔGD−N=−RTlnKD−N=−RTln[N][D],[1]where N is the soluble native structure, and D is the aggregation-competent unfolded state. Accordingly, it has been shown that the in vitro fibrillation of apoSOD1 displays the characteristic fingerprint of fragmentation-assisted growth (15) with a square root dependence on [D] (7), consistent with the requirement of sample agitation to expedite the reaction (1–4, 10). Analogous fibrillation behavior is found for β2-microglobulin (2), yeast prions Sup35 (16) and Ure2p (17), insulin (18), WW domain (19), TI 127 (20), and α-synuclein (21). The main difference between these proteins seems to be that some are intrinsically disordered and constantly aggregation-competent by lacking the ability to hide sticky sequence material by folding. In this study, we see that this simplistic in vitro behavior also translates to the more complex conditions in live tissue: the survival times of ALS mice expressing SOD1 variants of different stabilities are directly correlated with the in vivo levels of globally unfolded protein. Also, spinal cords of mice expressing the human SOD1 mutation G93A show exponential buildup of SOD1 aggregates with a square root dependence on log[D] indistinguishable from the fibrillation kinetics observed in agitated test tubes. The data raise fundamental questions about not only the striking resemblance between mouse and test tube aggregation but also, the apparent differences with human ALS pathology, which seems to have less ordered progression. Clues to the latter, however, are hinted in data from homozygous D90A mice showing two strains of structurally distinct SOD1 aggregates.Open in a separate windowFig. 1.SOD1 aggregation in vitro and in ALS mice. (A) Aggregation of SOD in test tubes yields fibrillar structures similar to those of other proteins (7). (B) Immunohistochemistry of the ventral horn in the terminal hSOD1^G93A mouse showing characteristics of aggresomes (44). (C) Competition between SOD1 folding and fibrillation in vitro, where elongation occurs by unfolded monomers through an encounter complex (7). The question that we ask is how do the in vitro and in vivo aggregations compare mechanistically. (D) Agitation-induced fibrillation in vitro with representative data from an SOD1 mutant in 0 (blue) and 5 M (red) urea with the associated statistics of τ_1/2 for repeated measures. To account for this statistical variation, we use the distribution average (Table S1). (E) Log plot of ν_max vs. τ_1/2 for all individual measures in this study showing uniform behavior of the various SOD1 mutants and a slope of one characteristic for exponential growth (1–4). ALS-associated SOD1 mutations examined in ALS mice (red) (Table S1), other ALS-associated mutations (blue) (Table S1), and SOD1 control mutations (black) (Table S1).     

Jugular venous `a'' wave in pulmonary hypertension: new insights from a Doppler echocardiographic study

Objective—To study the mechanisms underlying the dominant `a' wave seen in patients with primary pulmonary hypertension.

Design—Retrospective and prospective examination of the jugular venous pulse recording, flow in the superior vena cava, and Doppler echocardiographic studies.

Setting—A tertiary referral centre for both cardiac and pulmonary disease, with facilities for invasive and noninvasive investigation, and assessment for heart and heart-lung transplantation.

Patients—12 patients with primary pulmonary hypertension, most being considered for heart-lung transplantation.

Results—Two distinct patterns of venous pulse and superior vena caval flow were identified: a dominant `a' wave with no `v' wave, an absent or poorly developed `y' descent, and exclusively systolic downward flow in the superior vena cava (group 1, n = 8), and a dominant `v' wave, deep `y' descent and exclusively diastolic downward flow in the superior vena cava (group 2, n = 4). A comparison between the two groups showed age (mean (SD)) 42 (18) ν 36 (7) years, RR interval 700 (65) ν 740 (240) ms, left ventricular end diastolic dimension 3·6 (0·8) ν 3·2 (1·0) cm and end systolic dimension 2·1 (0·5) ν 2·3 (0·3) cm, right ventricular end diastolic dimension 2·6 (0·5) ν 2·8 (0·6) cm, and pressure drop between right ventricle and right atrium 60 (8) ν 70 (34) mm Hg to be similar. Duration of tricuspid regurgitation 520 (30) ν 420 (130) ms and the time interval of pulmonary closure to the end of the tricuspid regurgitant signal 140 (30) ν 110 (40) ms were longer in group 1 compared with group 2, whereas right ventricular filling time was much shorter 180 (70) ν 350 (130) ms. In seven patients from group 1, a single peak of forward tricuspid flow was present, but this pattern was seen in only one patient from group 2.

Conclusions—In patients with primary pulmonary hypertension, the apparent `a' wave seen in the venous pulse is, in fact, a summation wave. It is probably the result of large pressure changes that must underlie rapid acceleration and deceleration of blood across the tricuspid valve when the right ventricular filling time is short.

     

Spectroscopic identification and stability of the intermediate in the OH + HONO2 reaction

The reaction of nitric acid with the hydroxyl radical influences the residence time of HONO₂ in the lower atmosphere. Prior studies [Brown SS, Burkholder JB, Talukdar RK, Ravishankara AR (2001) J Phys Chem A 105:1605–1614] have revealed unusual kinetic behavior for this reaction, including a negative temperature dependence, a complex pressure dependence, and an overall reaction rate strongly affected by isotopic substitution. This behavior suggested that the reaction occurs through an intermediate, theoretically predicted to be a hydrogen-bonded OH–HONO₂ complex in a six-membered ring-like configuration. In this study, the intermediate is generated directly by the association of photolytically generated OH radicals with HONO₂ and stabilized in a pulsed supersonic expansion. Infrared action spectroscopy is used to identify the intermediate by the OH radical stretch (ν₁) and OH stretch of nitric acid (ν₂) in the OH–HONO₂ complex. Two vibrational features are attributed to OH–HONO₂: a rotationally structured ν₁ band at 3516.8 cm⁻¹ and an extensively broadened ν₂ feature at 3260 cm⁻¹, both shifted from their respective monomers. These same transitions are identified for OD–DONO₂. Assignments of the features are based on their vibrational frequencies, analysis of rotational band structure, and comparison with complementary high level ab initio calculations. In addition, the OH (v = 0) product state distributions resulting from ν₁ and ν₂ excitation are used to determine the binding energy of OH–HONO₂, D₀ ≤ 5.3 kcal·mol⁻¹, which is in good accord with ab initio predictions.     

Entanglement coupling in linear polymers demonstrated by networks crosslinked in States of strain

Linear 1,2-polybutadiene is crosslinked at 0° by γ-irradiation while strained in simple extension, with extension ratios from 1.3 to 2.0. During irradiation times up to several hours, entanglement slippage is slight, since the temperature is only slightly above the glass transition. Subsequently, samples are released and reach their equilibrium states of ease at room temperature. From the extension ratio at state of ease, the ratio of ν_x (effective network strands terminated by crosslinks introduced) to ν_N (effective network strands terminated by entanglements) is calculated by composite network theories of Flory and others; and from the extension ratios together with the modulus, measured at small extensions, ν_N is calculated explicitly. It appears that ν_x increases approximately proportional to irradiation time; ν_N is approximately independent of irradiation, and it corresponds to a molecular weight between effective entanglement loci of about 13,000. This figure, however, which is larger than that deduced from rheological properties of the uncrosslinked polymer, is subject to future downward correction for partial entrapment of the entanglements and other refinements.     

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the α-subunit of intracellular G proteins (Gαβγ). Kinetic analysis of transducin (G_tαβγ) activation shows that an intermediary R*·G_tαβγ·GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*·G protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a G_tα C-terminal peptide (GαCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal α-helix of G_tα (α5) and shows how α5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them – termed stable or S-interaction – matches the position of the GαCT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of GαCT and conserved E(D)RY and NPxxY(x)_5,6F regions of the GPCR. The alternative fit – termed intermediary or I-interaction – is distinguished by a tilt (42°) and rotation (90°) of α5 relative to the S-interaction and shows different α5 contacts with the NPxxY(x)_5,6F region and the second cytoplasmic loop of R*. From the 2 α5 interactions, we derive a “helix switch” mechanism for the transition of R*·G_tαβγ·GDP to the nucleotide-free R*·G protein complex that illustrates how α5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.     

Jugular venous `a' wave in pulmonary hypertension: new insights from a Doppler echocardiographic study

Objective—To study the mechanisms underlying the dominant `a'' wave seen in patients with primary pulmonary hypertension.Design—Retrospective and prospective examination of the jugular venous pulse recording, flow in the superior vena cava, and Doppler echocardiographic studies.Setting—A tertiary referral centre for both cardiac and pulmonary disease, with facilities for invasive and noninvasive investigation, and assessment for heart and heart-lung transplantation.Patients—12 patients with primary pulmonary hypertension, most being considered for heart-lung transplantation.Results—Two distinct patterns of venous pulse and superior vena caval flow were identified: a dominant `a'' wave with no `v'' wave, an absent or poorly developed `y'' descent, and exclusively systolic downward flow in the superior vena cava (group 1, n = 8), and a dominant `v'' wave, deep `y'' descent and exclusively diastolic downward flow in the superior vena cava (group 2, n = 4). A comparison between the two groups showed age (mean (SD)) 42 (18) ν 36 (7) years, RR interval 700 (65) ν 740 (240) ms, left ventricular end diastolic dimension 3·6 (0·8) ν 3·2 (1·0) cm and end systolic dimension 2·1 (0·5) ν 2·3 (0·3) cm, right ventricular end diastolic dimension 2·6 (0·5) ν 2·8 (0·6) cm, and pressure drop between right ventricle and right atrium 60 (8) ν 70 (34) mm Hg to be similar. Duration of tricuspid regurgitation 520 (30) ν 420 (130) ms and the time interval of pulmonary closure to the end of the tricuspid regurgitant signal 140 (30) ν 110 (40) ms were longer in group 1 compared with group 2, whereas right ventricular filling time was much shorter 180 (70) ν 350 (130) ms. In seven patients from group 1, a single peak of forward tricuspid flow was present, but this pattern was seen in only one patient from group 2.Conclusions—In patients with primary pulmonary hypertension, the apparent `a'' wave seen in the venous pulse is, in fact, a summation wave. It is probably the result of large pressure changes that must underlie rapid acceleration and deceleration of blood across the tricuspid valve when the right ventricular filling time is short.     

The transition state for folding of an outer membrane protein

Inspired by the seminal work of Anfinsen, investigations of the folding of small water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilize their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Here we use mutational analysis to describe the transition state involved in the reversible folding of the β-barrel membrane protein PhoPQ-activated gene P (PagP) from a highly disordered state in 10 M urea to a native protein embedded in a lipid bilayer. Analysis of the equilibrium stability and unfolding kinetics of 19 variants that span all eight β-strands of this 163-residue protein revealed that the transition-state structure is a highly polarized, partly formed β-barrel. The results provide unique and detailed insights into the transition-state structure for β-barrel membrane protein folding into a lipid bilayer and are consistent with a model for outer membrane protein folding via a tilted insertion mechanism.     

Dynamical roles of metal ions and the disulfide bond in Cu, Zn superoxide dismutase folding and aggregation

Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in neuronal death in amyotrophic lateral sclerosis. Each SOD1 monomer binds to 1 copper and 1 zinc ion and maintains its disulfide bond (Cys-57–Cys-146) in the reducing cytoplasm of cell. Mounting experimental evidence suggests that metal loss and/or disulfide reduction are important for initiating misfolding and aggregation of SOD1. To uncover the role of metals and the disulfide bond in the SOD1 folding, we systemically study the folding thermodynamics and structural dynamics of SOD1 monomer and dimer with and without metal binding and under disulfide-intact or disulfide-reduced environments in computational simulations. We use all-atom discrete molecular dynamics for sampling. Our simulation results provide dynamical evidence to the stabilizing role of metal ions in both dimer and monomer SOD1. The disulfide bond anchors a loop (Glu-49 to Asn-53) that contributes to the dimer interface. The reduction of the disulfide bond in SOD1 with metal ions depleted results in a flexible Glu-49–Asn-53 loop, which, in turn, disrupts dimer formation. Interestingly, the disulfide bond reduction does not affect the thermostability of monomer SOD1 as significantly as the metal ions do. We further study the structural dynamics of metal-free SOD1 monomers, the precursor for aggregation, in simulations and find inhomogeneous local unfolding of β-strands. The strands protected by the metal-binding and electrostatic loops are found to unfold first after metal loss, leading to a partially unfolded β-sheet prone to aggregation. Our simulation study sheds light on the critical role of metals and disulfide bond in SOD1 folding and aggregation.     

The power of the optimal asymptotic tests of composite statistical hypotheses

The easily computable asymptotic power of the locally asymptotically optimal test of a composite hypothesis, known as the optimal C(α) test, is obtained through a “double” passage to the limit: the number n of observations is indefinitely increased while the conventional measure ξ of the error in the hypothesis tested tends to zero so that ξ_nn^½ → τ ≠ 0. Contrary to this, practical problems require information on power, say β(ξ,n), for a fixed ξ and for a fixed n. The present paper gives the upper and the lower bounds for β(ξ,n). These bounds can be used to estimate the rate of convergence of β(ξ,n) to unity as n → ∞. The results obtained can be extended to test criteria other than those labeled C(α). The study revealed a difference between situations in which the C(α) test criterion is used to test a simple or a composite hypothesis. This difference affects the rate of convergence of the actual probability of type I error to the preassigned level α. In the case of a simple hypothesis, the rate is of the order of n^-½. In the case of a composite hypothesis, the best that it was possible to show is that the rate of convergence cannot be slower than that of the order of n^-½ ln n.     

Magnetocaloric effect and magnetic cooling near a field-induced quantum-critical point

The presence of a quantum-critical point (QCP) can significantly affect the thermodynamic properties of a material at finite temperatures T. This is reflected, e.g., in the entropy landscape S(T,r) in the vicinity of a QCP, yielding particularly strong variations for varying the tuning parameter r such as pressure or magnetic field B. Here we report on the determination of the critical enhancement of ∂S/∂B near a B-induced QCP via absolute measurements of the magnetocaloric effect (MCE), (∂T/∂B)_S and demonstrate that the accumulation of entropy around the QCP can be used for efficient low-temperature magnetic cooling. Our proof of principle is based on measurements and theoretical calculations of the MCE and the cooling performance for a Cu²⁺-containing coordination polymer, which is a very good realization of a spin-½ antiferromagnetic Heisenberg chain—one of the simplest quantum-critical systems.     

Subelliptic estimates for complexes

New results are announced linking properties of the symbol module and characteristic variety of a differential complex with test estimates near the characteristic variety of the type considered by Hörmander (½-estimate). The first result is the invariance of the test estimates under pseudo-differential change of coordinates, and this leads to the introduction of a normal form for the complex in the neighborhood of a Cohen-MacCauley point of the symbol module. If the characteristic variety V is a manifold near the Cohen-MacCauley point (x₀,ζ₀) with parametrizing functions p₁,...,p_q, where q is the codimension of the characteristic variety in the complexified contangent bundle, the matrix [Formula: see text] of Poisson brackets defines invariantly a Hermitian form Q on the normal space to V at (x₀,ζ₀) when the dp_ζ(x₀,ζ₀) are used as basis, and the test estimates are satisfied at the ith stage of the complex if sig. Q (signature of Q) is ≥ n - i + 1 (n the dimension of the base manifold) or rank Q - sig. Q ≥ i + 1. Finally, conditions are given in order that, on a manifold with smooth boundary, the associated boundary complexes satisfy the ½-estimate.     

From the Cover: Mutant superoxide dismutase 1-induced IL-1β accelerates ALS pathogenesis

ALS is a fatal motor neuron disease of adult onset. Neuroinflammation contributes to ALS disease progression; however, the inflammatory trigger remains unclear. We report that ALS–linked mutant superoxide dismutase 1 (SOD1) activates caspase-1 and IL-1β in microglia. Cytoplasmic accumulation of mutant SOD1 was sensed by an ASC containing inflammasome and antagonized by autophagy, limiting caspase-1–mediated inflammation. Notably, mutant SOD1 induced IL-1β correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1β or treatment with recombinant IL-1 receptor antagonist (IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and attenuated inflammatory pathology. These findings identify microglial IL-1β as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.