bluuebunny/crossref_metadata_2025 huggingface dataset:
Microwave ponderomotive forces in solid-state ionic plasmas
Authors: Booske J. H. , Cooper R. F. , Freeman S. A. , Rybakov K. I. , Semenov V. E.
Published: July 2002

Numerous observations have been reported in the literature of enhanced mass transport and solid-state reaction rates during microwave heating of a variety of ceramic, glass, and polymer materials. An explanation for these controversial observations has eluded researchers for over a decade. This paper describes a series of recent experimental and theoretical investigations that provide an explanation for these intriguing observations in terms of ponderomotive forces acting on mobile ionic species. The ponderomotive phenomenon, like its conventional-plasma analog, can be described in the continuum model limit by combining the continuity, Poisson’s, and transport equations. However, the solid-state plasma version typically manifests as a result of gradients in mobile charge mobility (e.g., near physical surfaces or interfaces), whereas the conventional plasma ponderomotive transport is typically a consequence of gradients in the radiation field intensity. Both cases can be captured in a single, general, mathematical articulation developed in terms of the mobile particle fluxes.
Ion dynamics in multiple electrostatic waves in a magnetized plasma. I. Coherent acceleration
Authors: Bénisti D. , Ram A. K. , Bers A.
Published: July 2002

A new phenomenon of coherent acceleration of ions by a discrete spectrum of electrostatic waves propagating perpendicularly to a uniform magnetic field is described. It allows the energization of ions whose initial energies correspond to a region of phase space that is below the chaotic domain. The ion orbits below the chaotic domain are described very accurately using a perturbation analysis to second order in the wave amplitudes. This analysis shows that the coherent acceleration takes place only when the wave spectrum contains at least two waves whose frequencies are separated by an amount close to an integer multiple of the cyclotron frequency. The way the ion energization depends on the wave numbers and wave amplitudes is also presented in detail using the results of the perturbation analysis.
A new acoustic-like mode in an unmagnetized dusty plasma
Authors: Avinash K. , Shukla P. K.
Published: July 2002

It is shown that because of the dependence of the plasma potential φ on the dust number density nd for given background plasma parameters and the dust grain radius, viz. φ=φ(nd), the dusty plasma is endowed with an effective temperature Teff which is proportional to dφ/dnd. The gradient of the effective pressure nd0Teff provides a restoring force for a new acoustic-like mode, which is referred to as the dust electro-acoustic (DEA) mode whose phase velocity equals (Teff/md)1/2, where md is the dust mass. The acoustic-like nature of the DEA mode is interpreted in terms of an effective screening (due to the dust charge fluctuations), called the “Coulomb screening,” with the screening length λc=(Teff/4πndqd2)1/2, where qd is the dust charge.
High quality molecular beam epitaxial growth on patterned GaAs substrates
Authors: Smith John Stephen , Derry Pamela L. , Margalit Shlomo , Yariv Amnon
Published: July 2002

In this letter we describe a procedure for high quality molecular beam epitaxy (MBE) growth over finely patterned GaAs substrates which is suitable for device fabrication requiring lateral definition of small (∼1–2 μm) dimension. This method was used for the fabrication of index guided laser arrays. Yields of individual lasers exceeded 90%, and thresholds were uniform to 10%. Temperature and flux ratio dependence of faceting during MBE growth over patterned substrates is shown for temperatures ranging from 580 to 700 °C and for As/Ga flux ratios from 1.4:1 to 4:1. The real index guided structure, which can be formed by a single MBE growth over a ridged substrate, is discussed. This technique should prove useful in the fabrication of devices which take advantage of unique features formed during regrowth by MBE.
Magnetic depth profiling and characterization of Fe-oxide films by Kerr rotation and spin polarized photoemission
Authors: Kay Eric , Sigsbee R. A. , Bona G. L. , Taborelli M. , Siegmann H. C.
Published: July 2002

The site occupancy and the valence valence state of Fe ions in a ferritelike sputtered thin film and its subsequent magnetic properties are well known to critically depend on sample preparation techniques. We show how measuring spin polarization of threshold photoelectrons having a nondestructive depth profiling capability of up to 100 Å can be used to gain detailed insight into site and valency information as a function of depth. In particular, we demonstrate that a reactively sputter deposited Fe-oxide film nominally shown to have crystallography data consistent with bulk Fe3O4 is uniform throughout, but in fact has no divalent Fe++ contributing to its overall magnetic properties. Furthermore, post-deposition oxidation of this film to nominally yield the γFe2O3 structure resulted in producing a nonmagnetic top layer followed by a Fe+++ containing magnetic layer very similar to the as-deposited layer, but definitely not γFe2O3. Simple Kerr rotation measurements confirmed the presence of the nonmagnetic top layer to about 200 Å depth consistent with earlier measurements by neutron reflection. The evidence points to film structures with a high concentration of nonbulklike Fe site defects in the as-sputtered and oxidized condition.
Interfacial properties of InP and phosphorus deposited at low temperature
Authors: Schachter R. , Olego D. J. , Baumann J. A. , Bunz L. A. , Raccah P. M. , Spicer W. E.
Published: July 2002

We are reporting on a large reduction in the density of states on the InP surface by a phosphorus overlayer. Phosphorus, which is deposited at low temperatures, is a high-resistivity amorphous semiconductor that provides chemical and physical continuity at the interface. The density of surface states, extracted from capacitance-voltage data on metal-insulator-capacitor diodes, is ∼1011/cm2 eV at 0.1 eV below the conduction-band minimum. The low density of states near the conduction band is interpreted as a reduction of phosphorus vacancies at the P-InP interface.
Photo-assisted anisotropic etching of phosphorus-doped polycrystalline silicon employing reactive species generated by a microwave discharge
Authors: Hayasaka N. , Okano H. , Sekine M. , Horiike Y.
Published: July 2002

An anisotropic etching of heavily phosphorus-doped polycrystalline silicon is achieved by protecting the etched sidewall with polymerized film which results from the reaction of chlorine species and methyl methacrylate. Chlorine species are generated by a microwave discharge in Cl2 in the portion separated from a reaction chamber with a laser beam irradiation system. The laser beam enhances the removal of the polymerized film on the illuminated surface except the sidewall. And the Si/SiO2 etch rate ratio is infinite in this system.
Photovoltaic quantum well infrared detector
Authors: Goossen K. W. , Lyon S. A. , Alavi K.
Published: July 2002

A photovoltaic infrared detector based on photoemission from a single modulation doped GaAs-AlGaAs quantum well is presented. The modulation doping provides a built-in field which allows unbiased operation. We show results from a device which is sensitive to 180 meV (7 μm wavelength) light, with a bandwidth of 20 meV, for which we estimate the quantum efficiency to be 1%. We demonstrate that the detector may be tuned to different wavelengths by varying the width of the well.
Room-temperature excitonic optical nonlinearities of molecular beam epitaxially grown ZnSe thin films
Authors: Peyghambarian N. , Park S. H. , Koch S. W. , Jeffery A. , Potts J. E. , Cheng H.
Published: July 2002

Large optical nonlinearities have been observed in molecular beam epitaxially grown thin films of ZnSe at room temperature and at T=150 K. A comparison with a plasma theory indicates that in both cases exciton screening is the dominating mechanism for the nonlinearity. The maximum nonlinear index per excited electron-hole pair at room temperature is comparable to that of bulk GaAs and GaAs-AlGaAs multiple quantum wells. The measured absorption and nonlinear index spectra agree quite well with our calculated values.
The unexpected landscape of
in vivo
somatic mutation in a human epithelial cell lineage
Authors: Colgin Lorel M. , Hackmann Alden F. M. , Emond Mary J. , Monnat Raymond J.
Published: July 2002

Few data exist on somatic mutation in the epithelial cell lineages that play a central role in human biology and disease. To delineate the “landscape” of somatic mutation in a human epithelial cell lineage, we determined the frequency and molecular nature of somatic mutations occurring
in vivo
in the X-linked
HPRT
gene of kidney tubular epithelial cells. Kidney epithelial mutants were frequent (range 0.5 to 4.2 × 10
−4
) and contained a high proportion of unreported
HPRT
base substitutions, −1-bp deletions and multiple mutations. This spectrum of somatic mutation differed from
HPRT
mutations identified in human peripheral blood T lymphocytes and from germ-line
HPRT
mutations identified in Lesch–Nyhan syndrome or hyperuricemia patients. Our results indicate that DNA damage and mutagenesis may have unusual or mechanistically interesting features in kidney tubular epithelium, and that somatic mutation may play a more important role in human kidney disease than has been previously appreciated.
Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from
Escherichia coli
Authors: Borgnia Mario J. , Agre Peter
Published: July 2002

A large family of membrane channel proteins selective for transport
of water (aquaporins) or water plus glycerol (aquaglyceroporins) has
been found in diverse life forms.
Escherichia coli
has
two members of this family—a water channel, AqpZ, and a glycerol
facilitator, GlpF. Despite having similar primary amino acid sequences
and predicted structures, the oligomeric state and solute selectivity
of AqpZ and GlpF are disputed. Here we report biochemical and
functional characterizations of affinity-purified GlpF and compare
it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged
(HA-GlpF) polypeptides encoded by a bicistronic construct were
expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers
during Ni-nitrilotriacetate affinity purification. Sucrose gradient
sedimentation analyses showed that the oligomeric state of octyl
glucoside-solubilized GlpF varies: low ionic strength favors subunit
dissociation, whereas Mg
2+
stabilizes tetrameric assembly.
Reconstitution of affinity-purified GlpF into proteoliposomes increases
glycerol permeability more than 100-fold and water permeability up to
10-fold compared with control liposomes. Glycerol and water
permeability of GlpF both occur with low Arrhenius activation energies
and are reversibly inhibited by HgCl
2
. Our studies
demonstrate that, unlike AqpZ, a water-selective stable tetramer,
purified GlpF exists in multiple oligomeric forms under nondenaturing
conditions and is highly permeable to glycerol but less well permeated
by water.
Solution NMR spectroscopy of [α-
15
N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence
Authors: Klein-Seetharaman J. , Reeves P. J. , Loewen M. C. , Getmanova E. V. , Chung J. , Schwalbe H. , Wright P. E. , Khorana H. G.
Published: July 2002

[α-
15
N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside–phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20°C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [
13
C]Ser-338/[
15
N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330–Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.
Mapping the early steps in the pH-induced conformational conversion of the prion protein
Authors: Alonso Darwin O. V. , DeArmond Stephen J. , Cohen Fred E. , Daggett Valerie
Published: July 2002

Under certain conditions, the prion protein (PrP) undergoes a
conformational change from the normal cellular isoform,
PrP
C
, to PrP
Sc
, an infectious isoform capable
of causing neurodegenerative diseases in many mammals. Conversion can
be triggered by low pH, and
in vivo
this appears to
take place in an endocytic pathway and/or caveolae-like domains. It
has thus far been impossible to characterize the conformational change
at high resolution by experimental methods. Therefore, to investigate
the effect of acidic pH on PrP conformation, we have performed 10-ns
molecular dynamics simulations of PrP
C
in water at neutral
and low pH. The core of the protein is well maintained at neutral pH.
At low pH, however, the protein is more dynamic, and the
sheet-like structure increases both by lengthening of the native
β-sheet and by addition of a portion of the N terminus to widen the
sheet by another two strands. The side chain of Met-129, a
polymorphic codon in humans associated with variant Creutzfeldt–Jakob
disease, pulls the N terminus into the sheet. Neutralization of Asp-178
at low pH removes interactions that inhibit conversion, which is
consistent with the Asp-178–Asn mutation causing human prion diseases.
All-optical processing with molecular switches
Authors: Raymo Françisco M. , Giordani Silvia
Published: July 2002

A gradual transition from electrical to optical networks is accompanying the rapid progress of telecommunication technology. The urge for enhanced transmission capacity and speed is dictating this trend. In fact, large volumes of data encoded on optical signals can be transported rapidly over long distances. Their propagation along specific routes across a communication network is ensured by a combination of optical fibers and optoelectronic switches. It is becoming apparent, however, that the interplay between the routing electrical stimulations and the traveling optical signals will not be able to support the terabit-per-second capacities that will be needed in the near future. Electrical inputs cannot handle the immense parallelism potentially possible with optical signals. Operating principles to control optical signals with optical signals must be developed. Molecular and supramolecular switches are promising candidates for the realization of innovative materials for information technology. Binary digits can be encoded in their chemical, electrical, or optical inputs and outputs to execute specific logic functions. We have developed a simple strategy to gate optical signals with optical signals by using a photoactive molecular switch. We have demonstrated that NAND, NOR, and NOT operations can be implemented exclusively with optical inputs and optical outputs coupling from one to three switching elements. Our remarkably simple approach to all-optical switching might lead to the development of a new generation of devices for digital processing and communication technology.
Gating the bacterial mechanosensitive channel MscL
in
vivo
Authors: Batiza Ann Finney , Kuo Mario Meng-Chiang , Yoshimura Kenjiro , Kung Ching
Published: July 2002

YggB and MscL are the major mechanosensitive channels in
Escherichia coli
, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998)
J. Biol. Chem.
273, 26670–26674] to show that downshock gates MscL
in vivo
even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify
in vivo
(Leu-19→Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET
+
, thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an ≈800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19→Cys MscL in the presence of YggB, but a downshock of only ≈400 mmol/kg is required in the absence of YggB. In parallel, Leu-19→Cys MscL, stretched open by large sustained suction in the presence of MTSET
+
in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating
in vivo
, and that residue 19 becomes exposed upon channel opening.
Comparison of the dynamics of substrate access channels in three cytochrome P450s reveals different opening mechanisms and a novel functional role for a buried arginine
Authors: Winn Peter J. , Lüdemann Susanna K. , Gauges Ralph , Lounnas Valère , Wade Rebecca C.
Published: July 2002

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.
Conformational pathways in the gating of
Escherichia coli
mechanosensitive channel
Authors: Kong Yifei , Shen Yufeng , Warth Tiffany E. , Ma Jianpeng
Published: July 2002

The pathway of the gating conformational transition of
Escherichia coli
mechanosensitive channel was simulated, using the recently modeled open and closed structures, by targeted molecular dynamics method. The transition can be roughly viewed as a four-stage process. The initial motion under a lower tension load is predominantly elastic deformation. The opening of the inner hydrophobic pore on a higher tension load takes place after the major expansion of the outer channel dimension. The hypothetical N-terminal S1 helical bundle has been confirmed to form the hydrophobic gate, together with the M1 helices. The sequential breaking of the tandem hydrophobic constrictions on the M1 and S1 helices makes the two parts of the gate strictly coupled, acting as a single gate. The simulation also revealed that there is no significant energetic coupling between the inner S1 bundle and the outer M2 transmembrane helices. The molten-globular-like structural features of the S1 bundle in its intermediate open states may account for the observed multiple subconductance states. Moreover, the intermediate open states of mechanosensitive channels are not symmetric, i.e., the opening does not follow iris-like motion, which sharply contrasts to the potassium channel KcsA.
Comparison of the early stages of forced unfolding for fibronectin type III modules
Authors: Craig David , Krammer André , Schulten Klaus , Vogel Viola
Published: July 2002

The structural changes accompanying stretch-induced early unfolding
events were investigated for the four type III fibronectin (FN-III)
modules, FN-III
7
, FN-III
8
, FN-III
9
,
and FN-III
10
by using steered molecular dynamics.
Simulations revealed that two main energy barriers, I and II, have to
be overcome to initiate unraveling of FN-III's tertiary structure. In
crossing the first barrier, the two opposing β-sheets of FN-III are
rotated against each other such that the β-strands of both β-sheets
align parallel to the force vector (aligned state). All further events
in the unfolding pathway proceed from this intermediate state. A second
energy barrier has to be overcome to break the first major cluster of
hydrogen bonds between adjacent β-strands. Simulations revealed that
the height of barrier I varied significantly among the four modules
studied, being largest for FN-III
7
and lowest for
FN-III
10
, whereas the height of barrier II showed little
variation. Key residues affecting the mechanical stability of FN-III
modules were identified. These results suggest that FN-III modules can
be prestretched into an intermediate state with only minor changes to
their tertiary structures. FN-III
10
, for example, extends
12 Å from the native “twisted” to the intermediate aligned
state, and an additional 10 Å from the aligned state to further
unfolding where the first β-strand is peeled away. The implications
of the existence of intermediate states regarding the elasticity of
fibrillar fibers and the stretch-induced exposure of cryptic sites are
discussed.
Overexpression of p27
Kip1
lengthens the G
1
phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells
Authors: Mitsuhashi Takayuki , Aoki Yoko , Eksioglu Yaman Z. , Takahashi Takao , Bhide Pradeep G. , Reeves Steven A. , Caviness Verne S.
Published: July 2002

We describe a mouse model in which p27
Kip1
transgene
expression is spatially restricted to the central nervous system
neuroepithelium and temporally controlled with doxycycline.
Transgene-specific transcripts are detectable within 6 h
of doxycycline administration, and maximum nonlethal expression
is approached within 12 h. After 18–26 h of transgene expression,
the G
1
phase of the cell cycle is estimated to increase
from 9 to 13 h in the neocortical neuroepithelium, the maximum
G
1
phase length attainable in this proliferative population
in normal mice. Thus our data establish a direct link between
p27
Kip1
and control of G
1
phase length in the
mammalian central nervous system and unveil intrinsic mechanisms that
constrain the G
1
phase length to a putative physiological
maximum despite ongoing p27
Kip1
transgene expression.
Rhesus expression in a green alga is regulated by CO
2
Authors: Soupene Eric , King Natalie , Feild Eithne , Liu Phillip , Niyogi Krishna K. , Huang Cheng-Han , Kustu Sydney
Published: July 2002

The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH
3
, we recently speculated that Rh proteins are biological gas channels for CO
2
. Like NH
3
, CO
2
differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the
RH1
gene in the photosynthetic microbe
Chlamydomonas reinhardtii
. Expression of
RH1
was high for cells grown in air supplemented with 3% CO
2
or shifted from air to high CO
2
(3%) for 3 h. Conversely,
RH1
expression was low for cells grown in air (0.035% CO
2
) or shifted from high CO
2
to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO
2
.
Dimerization of a selectin and its ligand stabilizes cell rolling and enhances tether strength in shear flow
Authors: Ramachandran Vishwanath , Yago Tadayuki , Epperson Terry Kay , Kobzdej Marcin M. A. , Nollert Matthias U. , Cummings Richard D. , Zhu Cheng , McEver Rodger P.
Published: July 2002

Selectins mediate rolling of leukocytes by rapid formation and dissociation of selectin–ligand bonds, which are assumed to require high mechanical strength to prevent premature dissociation by the forces applied in shear flow. This assumption is based largely on the observation that increasing wall shear stress increases only modestly the dissociation of transient leukocyte tethers on very low selectin densities. P-selectin binds to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. Both PSGL-1 and P-selectin are extended homodimers. We perfused transfected cells expressing wild-type dimeric PSGL-1 or a chimeric monomeric form of PSGL-1 on immobilized dimeric or monomeric forms of P-selectin. Cells expressing dimeric or monomeric PSGL-1 tethered to P-selectin at equivalent rates. However, cells expressing dimeric PSGL-1 established more stable rolling adhesions, which were more shear resistant and exhibited less fluctuation in rolling velocities. On low densities of dimeric P-selectin, increasing wall shear stress more rapidly increased transient tether dissociation of cells expressing monomeric PSGL-1 than dimeric PSGL-1. Tether dissociation on low densities of monomeric P-selectin was even more shear sensitive. We conclude that dimerization of both PSGL-1 and P-selectin stabilizes tethering and rolling, probably by increasing rebinding within a bond cluster. Because transient tethers may have more than one bond, the mechanical strength of selectin–ligand bonds is likely to be lower than initially estimated. Tether strength may rely more on bond clusters to distribute applied force.
Myosin VI is a processive motor with a large step size
Authors: Rock Ronald S. , Rice Sarah E. , Wells Amber L. , Purcell Thomas J. , Spudich James A. , Sweeney H. Lee
Published: July 2002

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.
The S4–S5 linker couples voltage sensing and activation of pacemaker channels
Authors: Chen Jun , Mitcheson John S. , Tristani-Firouzi Martin , Lin Monica , Sanguinetti Michael C.
Published: July 2002

Voltage-gated channels are normally opened by depolarization and
closed by repolarization of the membrane. Despite sharing significant
sequence homology with voltage-gated K
+
channels, the
gating of hyperpolarization-activated, cyclic-nucleotide-gated (HCN)
pacemaker channels has the opposite dependence on membrane potential:
hyperpolarization opens, whereas depolarization closes, these channels.
The mechanism and structural basis of the process that couples voltage
sensor movement to HCN channel opening and closing is not understood.
On the basis of our previous studies of a mutant HERG (human
ether-a-go-go-related gene) channel, we hypothesized that the
intracellular linker that connects the fourth and fifth transmembrane
domains (S4–S5 linker) of HCN channels might be important for channel
gating. Here, we used alanine-scanning mutagenesis of the HCN2 S4–S5
linker to identify three residues, E324, Y331, and R339, that when
mutated disrupted normal channel closing. Mutation of a basic residue
in the S4 domain (R318Q) prevented channel opening, presumably by
disrupting S4 movement. However, channels with R318Q and Y331S
mutations were constitutively open, suggesting that these channels can
open without a functioning S4 domain. We conclude that the S4–S5
linker mediates coupling between voltage sensing and HCN channel
activation. Our findings also suggest that opening of HCN and related
channels corresponds to activation of a gate located near the inner
pore, rather than recovery of channels from a C-type inactivated state.
Binding sites in
Escherichia coli
dihydrofolate reductase communicate by modulating the conformational ensemble
Authors: Pan Hong , Lee J. Ching , Hilser Vincent J.
Published: July 2002

To explore how distal mutations affect binding sites and how
binding sites in proteins communicate, an ensemble-based model of the
native state was used to define the energetic connectivities between
the different structural elements of
Escherichia coli
dihydrofolate reductase. Analysis of this model protein has allowed us
to identify two important aspects of intramolecular communication.
First, within a protein, pair-wise couplings exist that define the
magnitude and extent to which mutational effects propagate from the
point of origin. These pair-wise couplings can be identified from a
quantity we define as the residue-specific connectivity. Second, in
addition to the pair-wise energetic coupling between residues, there
exists functional connectivity, which identifies energetic coupling
between entire functional elements (i.e., binding sites) and the rest
of the protein. Analysis of the energetic couplings provides access to
the thermodynamic domain structure in dihydrofolate reductase as well
as the susceptibility of the different regions of the protein to both
small-scale (e.g., point mutations) and large-scale perturbations
(e.g., binding ligand). The results point toward a view of allosterism
and signal transduction wherein perturbations do not necessarily
propagate through structure via a series of conformational distortions
that extend from one active site to another. Instead, the observed
behavior is a manifestation of the distribution of states in the
ensemble and how the distribution is affected by the perturbation.
An optomechanical transducer in the blue light receptor phototropin from
Avena sativa
Authors: Salomon Michael , Eisenreich Wolfgang , Dürr Harald , Schleicher Erik , Knieb Elke , Massey Vincent , Rüdiger Wolfhart , Müller Franz , Bacher Adelbert , Richter Gerald
Published: July 2002

The
PHOT1
(
NPH1
) gene from
Avena sativa
specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine/threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of
A. sativa
phototropin with various
13
C/
15
N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by
1
H and
31
P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of
NPH1
.
A host–guest system to study structure–function relationships of membrane fusion peptides
Authors: Han Xing , Tamm Lukas K.
Published: July 2002

We designed a host–guest fusion peptide system, which is
completely soluble in water and has a high affinity for biological and
lipid model membranes. The guest sequences are those of the fusion
peptides of influenza hemagglutinin, which are solubilized by a highly
charged unstructured C-terminal host sequence. These peptides partition
to the surface of negatively charged liposomes or erythrocytes and
elicit membrane fusion or hemolysis. They undergo a conformational
change from random coil to an obliquely inserted (≈33° from the
surface) α-helix on binding to model membranes. Partition
coefficients for membrane insertion were measured for influenza fusion
peptides of increasing lengths (
n
= 8, 13, 16, and
20). The hydrophobic contribution to the free energy of binding of the
20-residue fusion peptide at pH 5.0 is −7.6 kcal/mol (1 cal =
4.18 J). This energy is sufficient to stabilize a “stalk”
intermediate if a typical number of fusion peptides assemble at the
site of membrane fusion. The fusion activity of the fusion peptides
increases with each increment in length, and this increase strictly
correlates with the hydrophobic binding energy and the angle of
insertion.
Three-helix-bundle protein in a Ramachandran model
Authors: Irbäck Anders , Sjunnesson Fredrik , Wallin Stefan
Published: July 2002

We study the thermodynamic behavior of a model protein with 54
amino acids that forms a three-helix bundle in its native state. The
model contains three types of amino acids and five to six atoms per
amino acid and has the Ramachandran torsional angles
φ

i

, ψ

i

as its
degrees of freedom. The force field is based on hydrogen bonds and
effective hydrophobicity forces. For a suitable choice of the relative
strength of these interactions, we find that the three-helix-bundle
protein undergoes an abrupt folding transition from an expanded state
to the native state. Also shown is that the corresponding one- and
two-helix segments are less stable than the three-helix sequence.
Identification of a trafficking determinant localized to the Kv1 potassium channel pore
Authors: Manganas Louis N. , Wang Qiang , Scannevin Robert H. , Antonucci Dana E. , Rhodes Kenneth J. , Trimmer James S.
Published: July 2002

The repertoire of Kv1 potassium channels expressed in presynaptic terminals of mammalian central neurons is shaped by intrinsic trafficking signals that determine surface-expression efficiencies of homomeric and heteromeric Kv1 channel complexes. Here, we show that a determinant controlling surface expression of Kv1 channels is localized to the highly conserved pore region. Point-mutation analysis revealed two residues as critical for channel trafficking, one in the extracellular “turret” domain and one in the region distal to the selectivity filter. Interestingly, these same residues also form the binding sites for polypeptide neurotoxins. Our findings demonstrate a previously uncharacterized function for the channel-pore domain as a regulator of channel trafficking.
Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability
Authors: Tehei Moeava , Madern Dominique , Pfister Claude , Zaccai Giuseppe
Published: July 2002

Protein thermal dynamics was evaluated by neutron scattering for
halophilic malate dehydrogenase from
Haloarcula
marismortui
(
Hm
MalDH) and BSA under different
solvent conditions. As a measure of thermal stability in each
case, loss of secondary structure temperatures were determined by CD.
Hm
MalDH requires molar salt and has different stability
behavior in H
2
O, D
2
O, and in NaCl and KCl
solvents. BSA remains soluble in molar NaCl. The neutron experiments
provided values of mean-squared atomic fluctuations at the 0.1 ns time
scale. Effective force constants, characterizing the mean resilience of
the protein structure, were calculated from the variation of the
mean-squared fluctuation with temperature. For
Hm
MalDH,
resilience increased progressively with increasing stability, from
molar NaCl in H
2
O, via molar KCl in D
2
O, to
molar NaCl in D
2
O. Surprisingly, however, the opposite was
observed for BSA; its resilience is higher in H
2
O where it
is less stable than in D
2
O. These results confirmed the
complexity of dynamics–stability relationships in different
proteins. Softer dynamics for BSA in D
2
O showed that the
higher thermostability is associated with entropic fluctuations. In the
halophilic protein, higher stability is associated with increased
resilience showing the dominance of enthalpic terms arising from
bonded interactions. From previous data, it is suggested that these are
associated with hydrated ion binding stabilizing the protein in the
high-salt solvent.
Coherent infrared emission from myoglobin crystals: An electric field measurement
Authors: Groot Marie-Louise , Vos Marten H. , Schlichting Ilme , van Mourik Frank , Joffre Manuel , Lambry Jean-Christophe , Martin Jean-Louis
Published: July 2002

We introduce coherent infrared emission interferometry as a χ
(2)
vibrational spectroscopy technique and apply it to studying the initial dynamics upon photoactivation of myoglobin (Mb). By impulsive excitation (using 11-fs pulses) of a Mb crystal, vibrations that couple to the optical excitation are set in motion coherently. Because of the order in the crystal lattice the coherent oscillations of the different proteins in the crystal that are associated with charge motions give rise to a macroscopic burst of directional multi-teraHertz radiation. This radiation can be detected in a phase-sensitive way by heterodyning with a broad-band reference field. In this way both amplitude and phase of the different vibrations can be obtained. We detected radiation in the 1,000–1,500 cm
−1
frequency region, which contains modes sensitive to the structure of the heme macrocycle, as well as peripheral protein modes. Both in carbonmonoxy-Mb and aquomet-Mb we observed emission from six modes, which were assigned to heme vibrations. The phase factors of the modes contributing to the protein electric field show a remarkable consistency, taking on values that indicate that the dipoles are created “emitting” at
t
= 0, as one would expect for impulsively activated modes. The few deviations from this behavior in Mb-CO we propose are the result of these modes being sensitive to the photodissociation process and severely disrupted by it.
Sequence-specific DNA-binding dominated by dehydration.
Authors: Lundbäck T , Härd T
Published: July 2002

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.
Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.
Authors: Bao J , Sharp A H , Wagster M V , Becher M , Schilling G , Ross C A , Dawson V L , Dawson T M
Published: July 2002

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.
Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.
Authors: Larsson O , Kindmark H , Brandstrom R , Fredholm B , Berggren P O
Published: July 2002

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).
Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.
Authors: Isenberg G , Etter E F , Wendt-Gallitelli M F , Schiefer A , Carrington W A , Tuft R A , Fay F S
Published: July 2002

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.
Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.
Authors: Henderson R M , Schneider S , Li Q , Hornby D , White S J , Oberleithner H
Published: July 2002

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.