Polar Loop Research Articles

Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag(+) or Cd(2+) on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag(+) and Cd(2+) sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag(+) or Cd(2+) chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0 with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H(+) translocation through F0 as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H(+) translocation to the cytoplasm.

A 28.3 mW PA-Closed Loop for Linearity and Efficiency Improvement Integrated in a $+$27.1 dBm WCDMA CMOS Power Amplifier

A low-cost, fully integrated CMOS PA is attractive. The main challenge is to improve its power efficiency. Although many linearization and efficiency improvement techniques have been proposed, most of these techniques are based on feed-forward, which may not be effective under varying PVT and load conditions [1–3]. The bandwidth is the issue for feedback techniques such as Cartesian and polar loop. There is a severe trade-off between output power and bandwidth, since generating high output power requires many gain stages including mixers and multiple drivers, which results in a large group delay. To overcome the trade-off, we propose a PA-closed loop, where an integrated feedback directly controls the final stage of a watt-level CMOS PA, as shown in Fig. 4.6.1. Although stable feedback at RF is difficult, separate phase and amplitude feedback can be realized without the stability issue. Large bandwidth and high output power are simultaneously realized, as the loop contains only the PA in the signal path and it is composed of a small number of building blocks. In addition, the proposed architecture is desirable for system integration, as the PA does not require an off-chip linearizer. The feedback improves ACLR with WCDMA output by 6dB, where the output power and PAE are +27.1dBm and 28%, respectively. Thanks to the feedback nature, it can improve the linearity and efficiency with varying load conditions, and is suitable for the increasing isolator-less applications. The linear output power and PAE are improved by up to 2.3dB and 6%, respectively, while the circuits for the loop consume 28.3mW.

NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by (1)H and (13)C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.

Fabrication of $\hbox{L}1_{2}\hbox{–}\hbox{CrPt}_{3}$ Alloy Films Using Rapid Thermal Annealing for Planar Bit Patterned Media

SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (2 nm)/Cr <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">25</sub> Pt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">75</sub> (15 nm) and SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (2 nm)/Cr <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">25</sub> Pt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">75</sub> (15 nm)/SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (10, 20 nm)/Co <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">80</sub> Zr <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">10</sub> Nb <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">10</sub> (10 nm) were prepared by magnetron sputtering method and post-annealed by rapid thermal annealing (RTA) at temperatures of 600°C-1000°C for 1-60 sec. The saturation magnetization M <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">s</sub> and coercivity H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">c</sub> measured by applying a maximum field of 18 kOe were 150 emu/cc and 12 kOe, respectively, for the sample after rapid thermal annealing at 1000 °C for 30 sec. This means that L1 <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> -CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> phase was obtained by RTA process. The RTA process was applied to fabricate the multilayered structure having L1 <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> -CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> and CoZrNb soft magnetic underlayer (SUL). The polar Kerr loop of CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> (15 nm)/SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (20 nm)/CoZrNb(10 nm) after RTA at 1000°C for 30 sec exhibited a large coercivity H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">c</sub> > 9 kOe corresponding to that of the CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> single layer. From the compositional depth profile of CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> (15 nm)/SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (20 nm)/CoZrNb(10 nm) processed by RTA at 1000°C for 30 sec, sharp layered structure was confirmed despite the high-temperature heat treatment. When the SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> thickness was reduced to 10 nm, such a layered structure was completely destroyed due to the interdiffusion between CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> and CoZrNb layers. Thus it was concluded that the RTA process and the interlayer of SiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (20 nm) were effective to fabricate layered structure having L1 <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> -CrPt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> and SUL.

Switching volume change and recording performance correlation for weakly coupled exchange spring media

We employed a new field sweep ratio dependence analysis method on the polar Kerr loop of exchange spring media to estimate the short time switching field, H0, and thermal stability factor, KUV/kBT, at various normalized Kerr signal levels, M, using modified Sharrock’s equation. For weakly coupled media, we observed that large switching volume changes around a “kink” in the Kerr loops. We observed a rapid increase in the KUV/kBT values before the kink, which suggests rapid switching of large areas of the soft layer via domain wall propagation. Small interfacial decoupling in exchange spring media between the soft and hard layers showed large degradation in media writability.

Structural Origin of Selectivity in Class II-Selective Histone Deacetylase Inhibitors

The development of class- and isoform-selective histone deacetylase (HDAC) inhibitors is highly desirable for the study of the complex interactions of these proteins central to transcription regulation as well as for the development of selective HDAC inhibitors as drugs in epigenetics. To provide a structural basis for the rational design of such inhibitors, a combined computational and experimental study of inhibition of three different histone deacetylase isoforms, HDAC1, -6, and -8, with three different hydroxamate inhibitors is reported. While SAHA was found to be unselective for the inhibition of class I and class II HDACs, the other inhibitors were found to be selective toward class II HDACs. Molecular dynamics simulations indicate that this selectivity is caused by both the overall shape of the protein surface leading to the active site and specific interactions of an aspartate residue in a polar loop and two phenylalanines and a methionine in a nonpolar loop. Monitoring the specific interactions as a function of the simulation time identifies a key sulfur-pi interaction. The implications of the structural motifs for the design of class II-selective HDAC inhibitors are discussed.

Structural Insight into AMPK Regulation: ADP Comes into Play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.

The rigid connecting loop stabilizes hairpin folding of the two helices of the ATP synthase subunit c

We have tested the role of the polar loop of subunit c of the Escherichia coli ATP synthase in stabilizing the hairpin structure of this protein. The structure of the c(32-52) peptide corresponding to the cytoplasmic region of subunit c bound to the dodecylphosphocholine micelles was solved by high-resolution NMR. The region comprising residues 41-47 forms a well-ordered structure rather similar to the conformation of the polar loop region in the solution structure of the full-length subunit c and is flanked by short alpha-helical segments. This result suggests that the rigidity of the polar loop significantly contributes to the stability of the hairpin formed by the two helices of subunit c. This experimental system may be useful for NMR studies of interactions between subunit c and subunits gamma and epsilon, which together form the rotor of the ATP synthase.

A ∼ 90° Late Silurian–Early Devonian apparent polar wander loop: The latest inertial interchange of planet earth?

Although rates of true polar wander have been small during the last 200 Myr of geological time primarily due to the slow response of the viscous lower mantle, it is possible for the Earth's rotation axis to move rapidly to the equator if mass perturbations within the interior cause the maximum and intermediate inertial axes to cross. Rotation of the silicate Earth is then recorded by a ∼ 90° arcuate sequence of palaeomagnetic poles embracing a time interval of < 20 Myr. An apparent polar wander loop (APW) of this length and duration is recorded by Silurian–Devonian palaeomagnetic data from two separated regions (the Caledonides and Gondwana) and is suggested by less well-constrained results from other continental blocks. The Caledonian and Gondwana data appear to identify a short term interchange of the Earth's inertial axes during the interval ∼ 410–390 Ma and yield a conventional continental reconstruction highlighting pending collision between Laurentia and the western margin of Gondwana near the Silurian–Devonian boundary to isolate the Rheic Ocean. This APW loop was accompanied by the extinction of successive oceans and occurred during, or shortly before, continental collisions between Gondwana, Laurentia, Baltica and Siberia to form the bulk of the supercontinent Pangaea. The implied rapid translation of the outer Earth resulting from transient modification of the Earth's figure is therefore most plausibly related to the avalanching of long lithosphere slabs into the lower mantle. The inertial interchange appears to be recorded by contemporaneous sea level changes although more refined stratigraphic data are required to confirm this.

Palaeomagnetism and40Ar/39Ar geochronology of mafic dykes from the eastern Bushveld Complex (South Africa)

SUMMARY We report palaeomagnetic and 40Ar/39Ar age data for dykes that intrude the ∼2 Ga eastern Bushveld Igneous Complex (BIC). The dykes were previously assumed to be of Karroo age (Jurassic ∼ 180 Ma) based on their NE–SW orientation. Palaeomagnetic data (pole position 8.7°N, 22°E; dp/dm= 18/20.6°), however, clearly demonstrate that these dykes are Precambrian in age, either ∼1.9 Ga and close to the Early Proterozoic Bushveld age, or 1649 ± 10 Ma based on 40Ar/39Ar plagioclase laser fusion ages from one of the dykes. Both normal and reverse polarity dykes are identified, and a positive reversal test together with a semi-conclusive contact test attests to a primary magnetization. If the 40Ar/39Ar age represents a primary cooling age then palaeomagnetic poles from South Africa (Kalahari) at ∼1.9 to ∼2 Ga and ∼1650 Ma are virtually identical, and suggest an apparent polar wander loop; alternatively, the Kalahari Craton drifted from high southerly (>50°) to high northerly latitudes (or vice versa) during this interval. Conversely, if we assign a ∼1.9 Ga age for dyke emplacement as suggested from a comparison with Kalahari palaeomagnetic poles (e.g. Waterberg–Soutpansberg pole), the ∼1650 Ma 40Ar/39Ar age must relate to a thermal disturbance that did not erase the primary magnetic signature.

Quad-band GSM/GPRS/EDGE polar loop transmitter

An enhanced data-rates for GSM evolution (EDGE) transmitter using a nonlinear GSM-type PA is presented. It is based on a novel polar loop architecture that employs separate feedback control of the amplitude and the phase of the output signal. With this approach, the problems with AM-to-PM as well as AM-to-AM of the nonlinear PA are essentially eliminated. In addition, this architecture allows for a large dynamic output power control range, as required by the GSM specification. The transmitter uses a standard I/Q interface and does not require the extraction of amplitude and phase modulation in the digital domain. The dual feedback loop ensures robust performance even under voltage-standing wave ratio variations without using an isolator. No external PA filtering is required to meet the transmitter noise in the receive band. The EDGE spectral mask is met with an rms error vector magnitude of <3% at 29 dBm at the antenna, corresponding to 2 dB above nominal maximum output power. There is no mode change between GMSK and EDGE, and the transmitter operates seamlessly in multislot Enhanced General Packet Radio Service. The polar modulation transmitter meets or exceeds the GSM-type approval requirements for both EDGE and GMSK in quad band (850/900/1800/1900 MHz).

Lysine 43 Is Trimethylated in Subunit c from Bovine Mitochondrial ATP Synthase and in Storage Bodies Associated with Batten Disease

The hydrophobic membrane protein, subunit c, has been isolated from ATP synthase purified from bovine heart mitochondria. It has also been obtained from lysosomal storage bodies associated with ceroid lipofuscinosis from ovine liver and from human brain tissue of a victim of Batten disease. It is likely that the lysosomal protein has originated from the mitochondrion. These samples have been characterized by mass spectrometric methods. Irrespective of its source, subunit c has an intact molecular mass of 7650 Da, 42 Da greater than the value calculated from the amino acid sequence, and the protein has been modified post-translationally. In all three samples, the modification is associated with lysine 43, which lies in a polar loop region linking the two transmembrane alpha-helices of the protein. This residue is conserved throughout vertebrate sequences. The additional mass arises from trimethylation and not acetylation at the epsilon-N-position of the residue. These experiments show that the post-translational modification of subunit c is not, as has been suggested, an abnormal phenomenon associated with the etiology of Batten disease and ceroid lipofucinoses. Evidently, it occurs either before or during import of the protein into mitochondria or at a mitochondrial location after completion of the import process. The function of the trimethyllysine residue in the assembled ATP synthase complex is obscure. The residue and the modification are not conserved in all ATP synthases, and their role in the assembly and (or) functioning of the enzyme appear to be confined to higher organisms.

Conformation of the gamma subunit at the gamma-epsilon-c interface in the complete Escherichia coli F(1)-ATPase complex by site-directed spin labeling.

Structure-function relationships of the gamma-epsilon-c subunit interface of F(O)F(1) ATP synthase, a region of subunit interactions important in coupling between catalysis and transport, were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The EPR line widths and collision accessibilities of 18 spin-labeled, unique cysteine F(1) mutants from gammaLeu198 to gammaLeu215 indicate an alternating pattern in the mobility and accessibility parameters for positions gamma201-209, which is reminiscent of a beta-strand. Labels at positions gamma204 and gamma210 show tertiary contact upon F(1) binding to F(O) and gammaD210C has reduced coupling efficiency. gammaE208C could not be spin labeled, but the uncoupling effects of gammaE208K are suppressed by second-site mutations in the polar loop of subunit c [Ketchum, C. J. and Nakamoto, R. K. (1998) J. Biol. Chem. 273, 22292-22297]. The restricted mobility and accessibility of spin labels in the odd-numbered positions between gamma201 and gamma207 plus the 2-4-fold higher values in k(cat) for ATP hydrolysis of these same mutant F(1) indicate that the interactions of these residues with the epsilon subunit mediate its inhibitory activity. Disrupted interactions with epsilon subunit also cause reduced coupling efficiency. We propose a model for the gamma-epsilon-c interface of Escherichia coli F(O)F(1) ATP synthase in which side chains from the odd-numbered residues of the gammaLys201-gammaTyr207 beta-strand directly and functionally interact with the epsilon subunit, while the even-numbered, acidic residues gammaAsp204, gammaGlu208, and gammaAsp210 interact with the F(O) sector, probably with subunit c. gamma Subunit interactions with both subunits in this region are important for coupling efficiency.

Increasing the talk-time of mobile radios with efficient linear transmitter architectures

Power efficiency of the RF transmitter stage in mobile handsets has a direct impact on battery capacity as well as talk-time between battery recharge cycles. Unfortunately the power amplifier (PA) linearisation methods in common usage today to meet the linearity specification of standards such as GSM EDGE, TETRA and UMTS result in low DC to RF efficiency. This paper discusses the relationship between linearity and transmitter efficiency and reviews various linearisation schemes: Cartesian loop, polar loop, 2nd harmonic injection, envelope elimination and restoration (EER), predistortion methods, and synthesis techniques (e.g. LINC and CALLUM). The paper also shows that to increase the power efficiency (and thus talk-time) hybrid solutions to power amplifier linearisation are required and these are also discussed.

Defining the Domain of Binding of F1 Subunit ε with the Polar Loop of F0 Subunit c in theEscherichia coli ATP Synthase

We have previously shown that the E31C-substituted epsilon subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 24609-24614). The interactions of subunits epsilon and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1. To further define the domains of interaction, we have introduced additional Cys into subunit epsilon and subunit c and tested for cross-link formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon, can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c. The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits. In the model, residues 26-33 form a turn of antiparallel beta-sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer.

Geochronology and palaeomagnetism of the Hunnedalen dykes, SW Norway: implications for the Sveconorwegian apparent polar wander loop

The post-Sveconorwegian Hunnedalen dyke swarm intrudes the high-grade Proterozoic gneiss complex of SW Norway. The dykes yield a Sm–Nd whole-rock-mineral date of 855±59 Ma and a 40Ar/ 39Ar biotite plateau date of 848±27 Ma (2 σ). The consistency of these two age determinations suggests that 850 Ma approximates the intrusion age of the swarm. Palaeomagnetic data from the dykes match palaeomagnetic directions obtained from Late Sveconorwegian massive-type anorthosites (932–929 Ma) in SW Norway. The collective palaeomagnetic data (mean pole: latitude = 43.7°S, longitude = 213.3°E, A 95=4.6°) have uniform magnetic polarity, and, when considered in the context of the new geochronologic data, require that remanence acquisition in Sveconorwegian rocks (massif-type anorthosites and gneissic basement) of SW Norway is, in fact, not Sveconorwegian in age, but rather younger and related to a regional metamorphic/hydrothermal feature at ca. 850 Ma. The new age data question the validity of a counter-clockwise Sveconorwegian apparent polar wander loop in the Baltic data-sets and therefore require a critical re-evaluation of Rodinia Supercontinent fits based on palaeomagnetic data.

The palaeomagnetism of (Mesoproterozoic) Eriksfjord Group red beds, South Greenland: multiphase remagnetization during the Gardar and Grenville episodes

The Eriksfjord Group comprises ∼3000 m of lavas and sediments rapidly deposited in a rift which developed within an Andean-type batholith in juxtaposition to the southern margin of the Laurentian Shield in South Greenland at ca. 1300 Ma. The lavas have been shown to preserve a detailed record of the geomagnetic field at the time of eruption, incorporating normal, reversed and transitional directions. This study has examined the magnetic properties of the intervening red sediments. They are found to possess a diagenetic remanence imparted by mediating fluids at later times. The impact of diagenesis is stratigraphically controlled: the base of the rift infill has magnetizations partially resident in magnetite which are either unstable to thermal cleaning or record a single polarity ‘B’ magnetization (D/I=284/67°, 31 samples, α95=5.5°, palaeopole at 244.1°E, 47.5°N, dp/dm=7.5/9.1°). This corresponds in polarity, and closely in direction, to remanence observed in mid-Gardar gabbro giant dykes and dyke swarms emplaced along the axis of the rift system at ca. 1160 Ma; the causative diagenetic magnetite appears to have grown from hydrothermal systems motivated by this magmatism in a sealed reservoir setting within the lower part of the rift infill. The Ilímaussaq alkaline igneous complex was emplaced into the southern extension of the rift at ca. 1130 Ma and possesses a dual polarity magnetization (D/I=327/81°, α95=6.4°, 10 sites). Eriksfjord lavas within the thermal aureole are overprinted to varying degrees by comparable magnetizations with steep inclinations. The mean pole position (283°E, 71°N, dp/dm=12/12°) lies near the apex of an apparent polar wander loop incorporating the Gardar Track (ca. 1300–1140 Ma) and the Keweenawan Track (ca. 1115–1050 Ma). Magnetizations in the Eriksfjord sedimentary succession have not been significantly reset by emplacement of the Ilímaussaq complex, but higher levels of the rift infill are dominated by an ‘A’ magnetization (D/I=305/34°, α95=4.3°, 57 samples, palaeopole at 202.1°E, 32.4°N, dp/dm=2.8/4.9°) resident in haematite. The pole position does not correspond with any part of the Gardar Track, but does correlate with the return Keweenawan Track at ca. 1090 Ma, close to the time of Grenville orogenesis along the bordering southeastern margin of the Laurentian Shield. This remanence is attributed to diagenesis during extensional tectonism linked to the collapse of the Grenville Orogen formerly sited 100–200 km to the south.

A Mutation in the Escherichia coliF0F1-ATP Synthase Rotor, γE208K, Perturbs Conformational Coupling between Transport and Catalysis

Cross-linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving gamma and epsilon subunits in F1 and subunit c in F0 (Watts, S. D., Tang, C., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 28341-28347). To assess the function of these interactions, we introduced random mutations in this region of the gamma subunit (gamma194-213). One mutation, gammaGlu-208 to Lys (gammaE208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the gammaE208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 degreesC. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was linear from 20 to 50 degreesC. Analysis of this plot revealed a significant increase in the activation energy of the catalytic transition state to a value very similar to soluble, epsilon subunit-inhibited F1 and suggested that the mutation blocked normal release of epsilon inhibition of ATP hydrolytic activity upon binding of F1 to F0. The difference in temperature dependence suggested that the gammaE208K mutation perturbed release of inhibition via a different mechanism than it did energy coupling. Suppressor mutations in the polar loop of subunit c restored ATP-dependent H+ pumping and transition state thermodynamic parameters close to wild-type values indicating that interactions between gamma and c subunits mediate release of epsilon inhibition and communication of coupling information.

Interactions between the F1 and F0 Parts in the Escherichia coli ATP Synthase: ASSOCIATIONS INVOLVING THE LOOP REGION OF C SUBUNITS

The N-ethylmaleimide reactivity of c subunits in Escherichia coli F1F0 ATP synthase (ECF1F0) isolated from five mutants, each with a cysteine at a different position in the polar loop region (positions 39, 40, 42, 43, and 44), has been investigated. The maleimide was found to react with Cys placed at positions 42, 43, and 44 but not at 39 or 40. All copies of the c subunit reacted similarly when the Cys was at position 43 or 44. In contrast, the Cys in the mutant cQ42C reacted as two classes, with 60% reacting relatively rapidly and 40% reacting at a rate 40-fold slower. After removing F1, all copies of the c subunit in this mutant reacted equally fast. Therefore, the slow class in the cQ42C mutant represents c subunits shielded by, and probably involved directly in, the interaction of the F0 with gamma and epsilon subunits of the F1 part. Based on the estimated stoichiometry of c subunits in the ECF1F0 complex, 4 or 5 c subunits are involved in this F1 interaction. N-Ethylmaleimide modification of all of the c subunits reduced ATPase activity by only 30% in ECF1F0 from mutant cQ42C. Modification of the more rapidly reacting class had little effect on ATP hydrolysis-driven proton translocation, and did not alter the DCCD inhibition of ATPase activity. However, as those c subunits involved in the F1 interaction became modified, DCCD inhibition was progressively lost, as was coupling between ATP hydrolysis and proton translocation.

A homology model of the Id-3 helix-loop-helix domain as a basis for structure-function predictions

The function of the dominant negative Id (inhibitor of differentiation) helix-loop-helix (HLH) proteins is to dimerize with, and prevent the DNA binding of basic HLH (bHLH) transcription factors. A three-dimensional homology model was constructed for the HLH domain of human Id3 based on the X-ray crystal structures of the E47, MyoD, and Max bHLH proteins. The model showed that, in contrast to bHLH proteins, Id proteins appear able to dimerize without DNA stabilization because of better packing of the hydrophobic core, and the absence of destabilizing polar loop residues and of repulsive positive charges in the monomer interface at the base of the four α-helix bundle. This prediction was tested by in vitro protein-binding experiments, which showed that Id3 did indeed self-associate. It also showed that the inability of Id proteins to bind DNA arises from the non-basic, poorly defined, random coil structure of the region corresponding to that responsible for bHLH DNA-binding. A model of the Id1 protein was constructed and revealed a potential site of charge-charge repulsion in the hypothetical homodimer interface that may explain its observed inability to form homodimers.