bluuebunny/crossref_metadata_2025 huggingface dataset:
Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM
Authors: Marszalek Piotr E. , Li Hongbin , Oberhauser Andres F. , Fernandez Julio M.
Published: July 2002

Under a stretching force, the sugar ring of polysaccharide molecules switches from the chair to the boat-like or inverted chair conformation. This conformational change can be observed by stretching single polysaccharide molecules with an atomic force microscope. In those early experiments, the molecules were stretched at a constant rate while the resulting force changed over wide ranges. However, because the rings undergo force-dependent transitions, an experimental arrangement where the force is the free variable introduces an undesirable level of complexity in the results. Here we demonstrate the use of force-ramp atomic force microscopy to capture the conformational changes in single polysaccharide molecules. Force-ramp atomic force microscopy readily captures the ring transitions under conditions where the entropic elasticity of the molecule is separated from its conformational transitions, enabling a quantitative analysis of the data with a simple two-state model. This analysis directly provides the physico-chemical characteristics of the ring transitions such as the width of the energy barrier, the relative energy of the conformers, and their enthalpic elasticity. Our experiments enhance the ability of single-molecule force spectroscopy to make high-resolution measurements of the conformations of single polysaccharide molecules under a stretching force, making an important addition to polysaccharide spectroscopy.
Rapid compaction during RNA folding
Authors: Russell Rick , Millett Ian S. , Tate Mark W. , Kwok Lisa W. , Nakatani Bradley , Gruner Sol M. , Mochrie Simon G. J. , Pande Vijay , Doniach Sebastian , Herschlag Daniel , Pollack Lois
Published: July 2002

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the
Tetrahymena
group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.
DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in
Xenopus
egg extracts
Authors: Waga Shou , Masuda Taro , Takisawa Haruhiko , Sugino Akio
Published: July 2002

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA
replication, repair, and cell-cycle checkpoint control in eukaryotic
cells. Although the requirement of other replicative DNA polymerases,
DNA polymerases α and δ (Polα and δ), for chromosomal DNA
replication has been well documented by genetic and biochemical
studies, the precise role, if any, of Polɛ in chromosomal DNA
replication is still obscure. Here we show, with the use of a cell-free
replication system with
Xenopus
egg extracts, that
Xenopus
Polɛ is indeed required for chromosomal DNA
replication. In Polɛ-depleted extracts, the elongation step of
chromosomal DNA replication is markedly impaired, resulting in
significant reduction of the overall DNA synthesis as well as
accumulation of small replication intermediates. Moreover, despite the
decreased DNA synthesis, excess amounts of Polα are loaded onto the
chromatin template in Polɛ-depleted extracts, indicative of the
failure of proper assembly of DNA synthesis machinery at the fork.
These findings strongly suggest that Polɛ, along with Polα and
Polδ, is necessary for coordinated chromosomal DNA replication in
eukaryotic cells.
The preferred stoichiometry of
c
subunits in the rotary motor sector of
Escherichia coli
ATP synthase is 10
Authors: Jiang Weiping , Hermolin Joe , Fillingame Robert H.
Published: July 2002

The stoichiometry of
c
subunits in the
H
+
-transporting F
o
rotary motor of ATP synthase
is uncertain, the most recent suggestions varying from 10 to 14. The
stoichiometry will determine the number of H
+
transported
per ATP synthesized and will directly relate to the P/O ratio of
oxidative phosphorylation. The experiments described here show that the
number of
c
subunits in functional complexes of
F
o
F
1
ATP synthase from
Escherichia
coli
can be manipulated, but that the preferred number is 10.
Mixtures of genetically fused cysteine-substituted trimers
(
c
3
) and tetramers
(
c
4
) of subunit
c
were
coexpressed and the
c
subunits crosslinked in the plasma
membrane. Prominent products corresponding to oligomers of
c
7
and
c
10
were
observed in the membrane and purified F
o
F
1
complex, indicating that the
c
10
oligomer
formed naturally. Oligomers larger than
c
10
were also observed in the membrane fraction of cells expressing
c
3
or
c
4
individually, or in cells coexpressing
c
3
and
c
4
together, but these larger oligomers
did not copurify with the functional F
o
F
1
complex and were concluded to be aberrant products of assembly in the
membrane.
Ligand binding and protein dynamics in neuroglobin
Authors: Kriegl Jan M. , Bhattacharyya Aninda J. , Nienhaus Karin , Deng Pengchi , Minkow Oleksandr , Nienhaus G. Ulrich
Published: July 2002

Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O
2
and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3–353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275–353 K) exhibits four sequential kinetic features: (
i
) geminate rebinding (
t
< 1 μs); (
ii
) extremely fast bimolecular exogenous ligand binding (10 μs <
t
< 1 ms) with a nontrivial temperature dependence; (
iii
) endogenous ligand binding (100 μs <
t
< 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (
iv
) displacement of the endogenous by the exogenous ligand (10 ms <
t
< 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.
The long-range supraorganization of the bacterial photosynthetic unit: A key role for PufX
Authors: Frese Raoul N. , Olsen John D. , Branvall Rikard , Westerhuis Willem H. J. , Hunter C. Neil , van Grondelle Rienk
Published: July 2002

Bacterial photosynthesis relies on the interplay between light harvesting and electron transfer complexes, all of which are located within the intracytoplasmic membrane. These complexes capture and transfer solar energy, which is used to generate a proton gradient. In this study, we identify one of the factors that determines the organization of these complexes. We undertook a comparison of the organization of the light-harvesting complex 1 (LH1)/reaction center (RC) cores in the LH2
−
mutant of
Rhodobacter sphaeroides
in the presence or absence of the PufX protein. From polarized absorption spectra on oriented membranes, we conclude that PufX induces a specific orientation of the reaction center in the LH1 ring, as well as the formation of a long-range regular array of LH1-RC cores in the photosynthetic membrane. From our data, we have constructed a precise model of how the RC is positioned within the LH1 ring relative to the long (orientation) axis of the photosynthetic membrane.
Does recombination improve selection on codon usage? Lessons from nematode and fly complete genomes
Authors: Marais Gabriel , Mouchiroud Dominique , Duret Laurent
Published: July 2002

Understanding the factors responsible for variations in mutation
patterns and selection efficacy along chromosomes is a prerequisite for
deciphering genome sequences. Population genetics models predict a
positive correlation between the efficacy of selection at a given locus
and the local rate of recombination because of Hill–Robertson effects.
Codon usage is considered one of the most striking examples that
support this prediction at the molecular level. In a wide range of
species including
Caenorhabditis elegans
and
Drosophila melanogaster
, codon usage is essentially
shaped by selection acting for translational efficiency. Codon usage
bias correlates positively with recombination rate in
Drosophila
, apparently supporting the hypothesis that
selection on codon usage is improved by recombination. Here we present
an exhaustive analysis of codon usage in
C. elegans
and
D. melanogaster
complete genomes. We show that in both
genomes there is a positive correlation between recombination rate and
the frequency of optimal codons. However, we demonstrate that in both
species, this effect is due to a mutational bias toward G and C bases
in regions of high recombination rate, possibly as a direct consequence
of the recombination process. The correlation between codon usage bias
and recombination rate in these species appears to be essentially
determined by recombination-dependent mutational patterns, rather than
selective effects. This result highlights that it is necessary to take
into account the mutagenic effect of recombination to understand the
evolutionary role and impact of recombination.
An alternative carotenoid-to-bacteriochlorophyll energy transfer pathway in photosynthetic light harvesting
Authors: Papagiannakis Emmanouil , Kennis John T. M. , van Stokkum Ivo H. M. , Cogdell Richard J. , van Grondelle Rienk
Published: July 2002

Blue and green sunlight become available for photosynthetic energy conversion through the light-harvesting (LH) function of carotenoids, which involves transfer of carotenoid singlet excited states to nearby (bacterio)chlorophylls (BChls). The excited-state manifold of carotenoids usually is described in terms of two singlet states, S
1
and S
2
, of which only the latter can be populated from the ground state by the absorption of one photon. Both states are capable of energy transfer to (B)Chl. We recently showed that in the LH1 complex of the purple bacterium
Rhodospirillum rubrum
, which is rather inefficient in carotenoid-to-BChl energy transfer, a third additional carotenoid excited singlet state is formed. This state, which we termed S*, was found to be a precursor on an ultrafast fission reaction pathway to carotenoid triplet state formation. Here we present evidence that S* is formed with significant yield in the LH2 complex of
Rhodobacter sphaeroides
, which has a highly efficient carotenoid LH function. We demonstrate that S* is actively involved in the energy transfer process to BChl and thus have uncovered an alternative pathway of carotenoid-to-BChl energy transfer. In competition with energy transfer to BChl, fission occurs from S*, leading to ultrafast formation of carotenoid triplets. Analysis in terms of a kinetic model indicates that energy transfer through S* accounts for 10–15% of the total energy transfer to BChl, and that inclusion of this pathway is necessary to obtain a highly efficient LH function of carotenoids.
IL-15 mimics T cell receptor crosslinking in the induction of cellular proliferation, gene expression, and cytotoxicity in CD8+memory T cells
Authors: Liu Kebin , Catalfamo Marta , Li Yu , Henkart Pierre A. , Weng Nan-ping
Published: July 2002

Generation of CD8+memory T cells requires antigenic stimulation through T cell receptor (TCR); however, maintenance of CD8+memory T cells seems to be mediated by cytokines, such as IL-15, in a TCR-independent manner. Compared with the TCR-induced activation, less is known about the mechanisms of IL-15 action. We report here a comparative and kinetic analysis of the responses of memory phenotype CD8+T cells to IL-15 or TCR (anti-CD3) stimulationin vitro. These two stimuli induce highly similar responses in memory phenotype CD8+T cells as measured by cellular proliferation, gene expression changes, synthesis of effector molecules (IFNγ, tumor necrosis factor β, granzyme B, and perforin), and induction of cytotoxicity. From 189 genes/expressed sequence tags (ESTs) whose expression changed in CD8+memory T cells after IL-15 and anti-CD3 stimulation identified by cDNA microarray analysis, 77% of the genes/ESTs exhibit a highly similar pattern of expression between IL-15 and anti-CD3-treated cells, and only 16% and 7% of the genes/ESTs are differentially expressed in response to IL-15 and anti-CD3 treatments, respectively. These results show that IL-15 and anti-CD3 stimulation induced remarkably similar gene expression and effector function. Thus, IL-15 acts not only as a crucial growth factor but also as an antigen-independent activator of effector functions for CD8+memory T cells.
Generalized transduction in
Streptomyces coelicolor
Authors: Burke Julie , Schneider David , Westpheling Janet
Published: July 2002

We report the isolation of generalized transducing phages for
Streptomyces
species able to transduce chromosomal
markers or plasmids between derivatives of
Streptomyces
coelicolor
, the principal genetic model system for this
important bacterial genus. We describe four apparently distinct phages
(DAH2, DAH4, DAH5, and DAH6) that are capable of transducing multiple
chromosomal markers at frequencies ranging from 10
−5
to
10
−9
per plaque-forming unit. The phages contain DNA
ranging in size from 93 to 121 kb and mediate linked transfer of
genetic loci at neighboring chromosomal sites sufficiently close to be
packaged within the same phage particle. The key to our ability to
demonstrate transduction by these phages was the establishment of
conditions expected to severely reduce superinfection killing during
the selection of transductants. The host range of these phages, as
measured by the ability to form plaques, extends to species as
distantly related as
Streptomyces
avermitilis
and
Streptomyces
verticillus
, which are among the most commercially
important species of this genus. Transduction of plasmid DNA between
S. coelicolor
and
S. verticillus
was
observed at frequencies of ≈10
−4
transductants per
colony-forming unit.
Evaluating conformational free energies: The colony energy and its application to the problem of loop prediction
Authors: Xiang Zhexin , Soto Cinque S. , Honig Barry
Published: July 2002

In this paper, we introduce a method to account for the shape of the potential energy curve in the evaluation of conformational free energies. The method is based on a procedure that generates a set of conformations, each with its own force-field energy, but adds a term to this energy that favors conformations that are close in structure (have a low rmsd) to other conformations. The sum of the force-field energy and rmsd-dependent term is defined here as the “colony energy” of a given conformation, because each conformation that is generated is viewed as representing a colony of points. The use of the colony energy tends to select conformations that are located in broad energy basins. The approach is applied to the
ab initio
prediction of the conformations of all of the loops in a dataset of 135 nonredundant proteins. By using an rmsd from a native criterion based on the superposition of loop stems, the average rmsd of 5-, 6-, 7-, and 8-residue long loops is 0.85, 0.92, 1.23, and 1.45 Å, respectively. For 8-residue loops, 60 of 61 predictions have an rmsd of less than 3.0 Å. The use of the colony energy is found to improve significantly the results obtained from the potential function alone. (The loop prediction program, “Loopy,” can be downloaded at
http://trantor.bioc.columbia.edu
.)
DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature
Authors: Askwith Candice C. , Benson Christopher J. , Welsh Michael J. , Snyder Peter M.
Published: July 2002

Several DEG/ENaC cation channel subunits are expressed in
the tongue and in cutaneous sensory neurons, where they are postulated
to function as receptors for salt and sour taste and for touch. Because
these tissues are exposed to large temperature variations, we examined
how temperature affects DEG/ENaC channel function. We found that
cold temperature markedly increased the constitutively active
Na
+
currents generated by epithelial Na
+
channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold
temperature did not induce current from other DEG/ENaC family
members (BNC1, ASIC, and DRASIC). However, when these channels were
activated by acid, cold temperature potentiated the currents by slowing
the rate of desensitization. Potentiation was abolished by a
“Deg” mutation that alters channel gating. Temperature changes in
the physiologic range had prominent effects on current in cells
heterologously expressing acid-gated DEG/ENaC channels, as well
as in dorsal root ganglion sensory neurons. The finding that cold
temperature modulates DEG/ENaC channel function may provide a
molecular explanation for the widely recognized ability of temperature
to modify taste sensation and mechanosensation.
Functional dynamics in the active site of the ribonuclease binase
Authors: Wang Lincong , Pang Yuxi , Holder Tina , Brender Jeffrey R. , Kurochkin Alexander V. , Zuiderweg Erik R. P.
Published: July 2002

Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-μs time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a β-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access/egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum
k
cat
(1,500 s
−1
) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover.
Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation
Authors: Spörlein Sebastian , Carstens Heiko , Satzger Helmut , Renner Christian , Behrendt Raymond , Moroder Luis , Tavan Paul , Zinth Wolfgang , Wachtveitl Josef
Published: July 2002

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical
cis
/
trans
-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.
Polarity and permeation profiles in lipid membranes
Authors: Marsh Derek
Published: July 2002

The isotropic
14
N-hyperfine coupling constant,
a

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, of nitroxide spin labels is dependent on the local environmental polarity. The dependence of
a

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in fluid phospholipid bilayer membranes on the C-atom position,
n
, of the nitroxide in the
sn
-2 chain of a spin-labeled diacyl glycerophospholipid therefore determines the transmembrane polarity profile. The polarity variation in phospholipid membranes, with and without equimolar cholesterol, is characterized by a sigmoidal, trough-like profile of the form {1 + exp [(
n
−
n
o
)/λ]}
−1
, where
n
=
n
o
is the point of maximum gradient, or polarity midpoint, beyond which the free energy of permeation decreases linearly with
n
, on a characteristic length-scale, λ. Integration over this profile yields a corresponding expression for the permeability barrier to polar solutes. For fluid membranes without cholesterol,
n
o
≈ 8 and λ ≈ 0.5–1 CH
2
units, and the permeability barrier introduces an additional diffusive resistance that is equivalent to increasing the effective membrane thickness by 35–80%, depending on the lipid. For membranes containing equimolar cholesterol,
n
o
≈ 9–10, and the total change in polarity is greater than for membranes without cholesterol, increasing the permeability barrier by a factor of 2, whereas the decay length remains similar. The permeation of oxygen into fluid lipid membranes (determined by spin-label relaxation enhancements) displays a profile similar to that of the transmembrane polarity but of opposite sense. For fluid membranes without cholesterol
n
o
≈ 8 and λ ≈ 1 CH
2
units, also for oxygen. The permeation profile for polar paramagnetic ion complexes is closer to a single exponential decay, i.e.,
n
o
lies outside the acyl-chain region of the membrane. These results are relevant not only to the permeation of water and polar solutes into membranes and their permeabilities, but also to depth determinations of site-specifically spin-labeled protein residues by using paramagnetic relaxation agents.
Three SNARE complexes cooperate to mediate membrane fusion
Authors: Hua Yuanyuan , Scheller Richard H.
Published: July 2002

Soluble
N-
ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the syntaxin, SNAP-25, and VAMP families mediate intracellular membrane fusion through the formation of helical bundles that span opposing membranes. Soluble SNARE domains that lack their integral membrane anchors inhibit membrane fusion by forming nonfunctional complexes with endogenous SNARE proteins. In this study we investigate the dependence of membrane fusion on the concentration of a soluble SNARE coil domain derived from VAMP2. The increase in the inhibition of fusion observed with increasing concentration of inhibitor is best fit to a function that suggests three SNARE complexes cooperate to mediate fusion of a single vesicle. These three complexes likely contribute part of a protein and lipidic fusion pore.
Positive cooperativity without domains or subunits in a monomeric membrane channel
Authors: Rostovtseva Tatiana K. , Liu Tong-Tong , Colombini Marco , Parsegian V. Adrian , Bezrukov Sergey M.
Published: July 2002

The monomeric VDAC channel shows an accelerated pH titration of its transport properties with a Hill coefficient of about 2. This manifests itself as a sharp peak in conductance noise as well as a fast change in channel selectivity with pH. On the basis of the known structure of this channel, we propose that this cooperativity arises from a mechanically linked mobile pair of ionizable groups. Concerted movement of these groups between two states changes the distance from nearby electrostatic charge to influence the pK of the groups. This model of pH-dependent motion produces positive cooperative behavior that fits the observations without need for subunits or identifiable domains within the protein. The mathematical formalism has never required such domains, but these are generally considered an essential part of cooperative behavior in proteins. The present proposal reduces the size of a cooperative unit to a minimum, extending the limits of what is perceived to be possible. Together with large-scale conformational transitions, these subtle cooperative structural changes may allow proteins to adapt, with high sensitivity, to changes in their environment. They might also be relatively easy to engineer into a protein.
Genetic control of resistance to experimental infection with virulentMycobacterium tuberculosis
Authors: Kramnik Igor , Dietrich William F. , Demant Peter , Bloom Barry R.
Published: July 2002

Over 2 billion people are estimated to be infected with virulentMycobacterium tuberculosis, yet fewer than 10% progress to clinical tuberculosis within their lifetime. Twin studies and variations in the outcome of tuberculosis infection after exposure to similar environmental risks suggest genetic heterogeneity among individuals in their susceptibility to disease. In a mouse model of tuberculosis, we have established that resistance and susceptibility to virulentM. tuberculosisis a complex genetic trait. A new locus with a major effect on tuberculosis susceptibility, designatedsst1(susceptibility to tuberculosis 1), was mapped to a 9-centimorgan (cM) interval on mouse chromosome 1. It is located 10–19 cM distal to a previously identified gene,Nramp1, that controls the innate resistance of mice to the attenuated bacillus Calmette–Guérin vaccine strain. The phenotypic expression of the newly identified locus is distinct from that ofNramp1in thatsst1controls progression of tuberculosis infection in a lung-specific manner. Mice segregating at thesst1locus exhibit marked differences in the growth rates of virulent tubercle bacilli in the lungs. Lung lesions in congenicsst1-susceptible mice are characterized by extensive necrosis and unrestricted extracellular multiplication of virulent mycobacteria, whereassst1-resistant mice develop interstitial granulomas and effectively control multiplication of the bacilli. The resistant allele ofsst1,although powerful in controlling infection, is not sufficient to confer full protection against virulentM. tuberculosis, indicating that other genes located outside of thesst1locus are likely also to be important for controlling tuberculosis infection.
Kinetic trapping of DNA by transcription factor IIIB
Authors: Cloutier Timothy E. , Librizzi Monett D. , Mollah A. K. M. M. , Brenowitz Michael , Willis Ian M.
Published: July 2002

High levels of RNA polymerase III gene transcription are achieved
by facilitated recycling of the polymerase on transcription factor IIIB
(TFIIIB)-DNA complexes that are stable through multiple rounds of
initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding
protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The
high stability of the TFIIIB-DNA complex is conferred by TFIIIB90
binding to TFIIIB70-TBP-DNA complexes. This stability is thought to
result from compound bends introduced in the DNA by TBP and TFIIIB90
and by protein–protein interactions that obstruct DNA dissociation.
Here we present biochemical evidence that the high stability of
TFIIIB-DNA complexes results from kinetic trapping of the DNA.
Thermodynamic analysis shows that the free energies of formation of
TFIIIB70-TBP-DNA (Δ
G
° = −12.10 ± 0.12
kcal/mol) and TFIIIB-DNA (Δ
G
° = −11.90 ±
0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis
shows that the half-lives of these complexes (46 ± 3 min and
95 ± 6 min, respectively) differ significantly. The differential
stability of these isoenergetic complexes demonstrates that TFIIIB90
binding energy is used to drive conformational changes and increase the
barrier to complex dissociation.
Periodic perturbations in
Shaker
K
+
channel gating kinetics by deletions in the S3–S4 linker
Authors: Gonzalez Carlos , Rosenman Eduardo , Bezanilla Francisco , Alvarez Osvaldo , Latorre Ramon
Published: July 2002

Upon depolarization positive charges contained in the transmembrane
segment S4 of voltage-dependent channels are displaced from the
cytoplasmic to the external milieu. This charge movement leads to
channel opening. In
Shaker
K
+
channels four
positively charged arginines in the S4 domain are transferred from the
internal to the external side of the channel during activation. The
distance traveled by the S4 segment during activation is unknown, but
large movements should be constrained by the S3–S4 linker.
Constructing deletion mutants, we show that the activation time
constant and the midpoint of the voltage activation curve of the
Shaker
K
+
channel macroscopic currents
becomes a periodic function of the S3–S4 linker length for linkers
shorter than 7 aa residues. The periodicity is that typical of
α-helices. Moreover, a linker containing only 3 aa is enough to
recover the wild-type phenotype. The deletion method revealed the
importance of the S3–S4 linker in determining the channel gating
kinetics and indicated that the α-helical nature of S4 extends toward
its N terminus. These results support the notion that a small
displacement of the S4 segment suffices to displace the four gating
charges involved in channel opening.
DsbC activation by the N-terminal domain of DsbD
Authors: Goldstone David , Haebel Peter W. , Katzen Federico , Bader Martin W. , Bardwell James C. A. , Beckwith Jon , Metcalf Peter
Published: July 2002

The correct formation of disulfide bonds in the periplasm of
Escherichia coli
involves Dsb proteins, including two
related periplasmic disulfide-bond isomerases, DsbC and DsbG. DsbD is a
membrane protein required to maintain the functional oxidation state of
DsbC and DsbG. In this work, purified proteins were used to investigate
the interaction between DsbD and DsbC. A 131-residue N-terminal
fragment of DsbD (DsbDα) was expressed and purified and shown to form
a functional folded domain. Gel filtration results indicate that
DsbDα is monomeric. DsbDα was shown to interact directly with and
to reduce the DsbC dimer, thus increasing the isomerase activity of
DsbC. The DsbC–DsbDα complex was characterized, and formation of the
complex was shown to require the N-terminal dimerization domain of
DsbC. These results demonstrate that DsbD interacts directly with
full-length DsbC and imply that no other periplasmic components are
required to maintain DsbC in the functional reduced state.
Genetic engineering of phytochrome biosynthesis in bacteria
Authors: Gambetta Gregory A. , Lagarias J. Clark
Published: July 2002

The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.
High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking
Authors: Kues Thorsten , Dickmanns Achim , Lührmann Reinhard , Peters Reiner , Kubitscheck Ulrich
Published: July 2002

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are components of the splicing machinery that removes introns from precursor mRNA. Like other splicing factors, U snRNPs are diffusely distributed throughout the nucleus and, in addition, are concentrated in distinct nuclear substructures referred to as speckles. We have examined the intranuclear distribution and mobility of the splicing factor U1 snRNP on a single-molecule level. Isolated U1 snRNPs were fluorescently labeled and incubated with digitonin-permeabilized 3T3 cells in the presence of
Xenopus
egg extract. By confocal microscopy, U1 snRNPs were found to be imported into nuclei, yielding a speckled intranuclear distribution. Employing a laser video-microscope optimized for high sensitivity and high speed, single U1 snRNPs were visualized and tracked at a spatial precision of 35 nm and a time resolution of 30 ms. The single-particle data revealed that U1 snRNPs occurred in small clusters that colocalized with speckles. In the clusters, U1 snRNPs resided for a mean decay time of 84 ms before leaving the optical slice in the direction of the optical axis, which corresponded to a mean effective diffusion coefficient of 1 μm
2
/s. An analysis of the trajectories of single U1 snRNPs revealed that at least three kinetic classes of low, medium, and high mobility were present. Moreover, the mean square displacements of these fractions were virtually independent of time, suggesting arrays of binding sites. The results substantiate the view that nuclear speckles are not rigid structures but highly dynamic domains characterized by a rapid turnover of U1 snRNPs and other splicing factors.
An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel β-fibrils
Authors: Bevivino Anthony E. , Loll Patrick J.
Published: July 2002

The protein ataxin-3 contains a polyglutamine region; increasing the number of glutamines beyond 55 in this region gives rise to the neurodegenerative disease spinocerebellar ataxia type 3. This disease and other polyglutamine expansion diseases are characterized by large intranuclear protein aggregates (nuclear inclusions). By using full-length human ataxin-3, we have investigated the changes in secondary structure, aggregation behavior, and fibril formation associated with an increase from the normal length of 27 glutamines (Q27 ataxin-3) to a pathogenic length of 78 glutamines (Q78 ataxin-3). Q78 ataxin-3 aggregates strongly and could be purified only when expressed with a solubility-enhancing fusion-protein partner. A marked decrease in α-helical secondary structure accompanies expansion of the polyglutamine tract, suggesting destabilization of the native protein. Proteolytic removal of the fusion partner in the Q78 protein, but not in the Q27 protein, leads to the formation of SDS-resistant aggregates and Congo-red reactive fibrils. Infrared spectroscopy of fibrils reveals a high β-sheet content and suggests a parallel, rather than an antiparallel, sheet conformation. We present a model for a polar zipper composed of parallel polyglutamine β-sheets. Our data show that intact ataxin-3 is fully competent to form aggregates, and posttranslational cleavage or other processing is not necessary to generate a misfolding event. The data also suggest that the protein aggregation phenotype associated with glutamine expansion may derive from two effects: destabilization of the native protein structure and an inherent propensity for β-fibril formation on the part of glutamine homopolymers.
Formation of supramolecular activation clusters on freshex vivoCD8+T cells after engagement of the T cell antigen receptor and CD8 by antigen-presenting cells
Authors: Potter Terry A. , Grebe Kristie , Freiberg Ben , Kupfer Abraham
Published: July 2002

Upon productive interaction of CD4 T cells with antigen-presenting cells (APCs), receptors and intracellular proteins translocate and form spatially segregated supramolecular activation clusters (SMACs). It is not known whether SMACs are required for CD8 T cell activation. CD8 T cells, unlike CD4 T cells, can be activated by a single peptide-MHC molecule, or by purified monovalent recombinant peptide-MHC molecules. We studied, by three-dimensional digital microscopy, cell conjugates of freshex vivoCD8 T cells (obtained from OT-1 mice, which are transgenic for T cell antigen receptor reactive with the complex of H-2Kband the ovalbumin octapeptide SIINFEKL) and peptide-pulsed APCs. Remarkably, even in T cell:APC conjugates that were formed in the presence of the lowest concentration of peptide that was sufficient to elicit T cell proliferation and IFN-γ production; the θ isoform of protein kinase C was clustered in a central SMAC, and lymphocyte function-associated antigen 1 and talin were clustered in the peripheral SMAC. Conjugation of T cells to APCs that were pulsed with concentrations of peptide smaller than that required to activate T cells was greatly reduced, and SMACs were not formed at all. APCs expressing mutant H-2Kb(Lys227) molecules that do not bind CD8 were unable to form stable conjugates with these T cells, even at high peptide concentrations. Thus, although CD8 and CD4 T cells may display different sensitivity to the concentration and oligomerization of surface receptors, SMACs are formed and seem to be required functionally in both cell types. However, unlike CD4 T cells, which can form SMACs without CD4, CD8 T cells from OT-1 transgenic mice depend on their coreceptor, CD8, for the proper formation of SMACs .
Oligomerization of the integrin αIIbβ3: Roles of the transmembrane and cytoplasmic domains
Authors: Li Renhao , Babu Charles R. , Lear James D. , Wand A. Joshua , Bennett Joel S. , DeGrado William F.
Published: July 2002

Integrins are a family of α/β heterodimeric membrane proteins, which mediate cell–cell and cell–matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the α and β subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of αIIbβ3 [Hughes, P. E.,
et al.
(1996)
J. Biol. Chem.
271, 6571–6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the α and β subunits of αIIbβ3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the α- and β-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.
Cholesterol depletion induces large scale domain segregation in living cell membranes
Authors: Hao Mingming , Mukherjee Sushmita , Maxfield Frederick R.
Published: July 2002

Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents.
A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR)
Authors: Chan Ting-Fung , Carvalho John , Riles Linda , Zheng X. F. Steven
Published: July 2002

The target of rapamycin protein (TOR) is a highly conserved
ataxia telangiectasia-related protein kinase essential for cell growth.
Emerging evidence indicates that TOR signaling is highly complex and is
involved in a variety of cellular processes. To understand its general
functions, we took a chemical genomics approach to explore the genetic
interaction between TOR and other yeast genes on a genomic scale. In
this study, the rapamycin sensitivity of individual deletion mutants
generated by the
Saccharomyces
Genome Deletion Project
was systematically measured. Our results provide a global view of the
rapamycin-sensitive functions of TOR. In contrast to conventional
genetic analysis, this approach offers a simple and thorough analysis
of genetic interaction on a genomic scale and measures genetic
interaction at different possible levels. It can be used to study the
functions of other drug targets and to identify novel protein
components of a conserved core biological process such as DNA damage
checkpoint/repair that is interfered with by a cell-permeable
chemical compound.
Pause and rotation of F
1
-ATPase during catalysis
Authors: Hirono-Hara Yoko , Noji Hiroyuki , Nishiura Masaya , Muneyuki Eiro , Hara Kiyotaka Y. , Yasuda Ryohei , Kinosita Kazuhiko , Yoshida Masasuke
Published: July 2002

F
1
-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central γ subunit relative to the surrounding α
3
β
3
ring. Here, we show that the rotation of F
1
-ATPase spontaneously lapses into long (≈30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F
1
-ATPase previously known as the ADP-Mg inhibited form in which F
1
-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the γ subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position.
Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum
Authors: Todorow Zlatko , Spang Anne , Carmack Edwin , Yates John , Schekman Randy
Published: July 2002

Mnn9p is a component of two distinct multiprotein complexes
in the
Saccharomyces cerevisiae cis-
Golgi that have both
been shown to have α-1,6-mannosyltransferase activity
in
vitro
. In one of these complexes, Mnn9p associates with four
other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the
other complex consists of Mnn9p and Van1p. Members of the
Mnn9p-containing complexes were incorporated into COPII vesicles made
in vitro
from endoplasmic reticulum (ER) membranes
isolated from cycloheximide-treated cells. This behavior is consistent
with an active Golgi to ER recycling process. To examine this path
in vivo
, we monitored retrograde transport of subunits
of the complex in cells blocked in anterograde transport from the ER.
In this situation, specific relocation of the proteins from the Golgi
to the ER was observed in the absence of new protein synthesis.
Conversely, when retrograde transport was blocked
in
vivo
, subunits of the mannosyltransferase complex accumulated
in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from
purified Golgi membranes was also investigated using a
coatomer-dependent vesicle budding assay. Gradient fractionation
experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were
incorporated into COPI-coated vesicles. These observations indicate
that the Mnn9p-containing mannosyltransferase complexes cycle back and
forth between the ER and Golgi.
Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.
Authors: Thornton A M , Buller R M , DeVico A L , Wang I M , Ozato K
Published: July 2002

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.
Single neuron control over a complex motor program.
Authors: Frost W N , Katz P S
Published: July 2002

While there are many instances of single neurons that can drive rhythmic stimulus-elicited motor programs, such neurons have seldom been found to be necessary for motor program function. In the isolated central nervous system of the marine mollusc Tritonia diomedea, brief stimulation (1 sec) of a peripheral nerve activates an interneuronal central pattern generator that produces the long-lasting (approximately 30-60 sec) motor program underlying the animal's rhythmic escape swim. Here, we identify a single interneuron, DRI (for dorsal ramp interneuron), that (i) conveys the sensory information from this stimulus to the swim central pattern generator, (ii) elicits the swim motor program when driven with intracellular stimulation, and (iii) blocks the depolarizing "ramp" input to the central pattern generator, and consequently the motor program itself, when hyperpolarized during the nerve stimulus. Because most of the sensory information appears to be funneled through this one neuron as it enters the pattern generator, DRI presents a striking example of single neuron control over a complex motor circuit.