bluuebunny/crossref_metadata_2025 huggingface dataset:
Product dependence and bifunctionality compromise the ultrasensitivity of signal transduction cascades
Authors: Ortega Fernando , Acerenza Luis , Westerhoff Hans V. , Mas Francesc , Cascante Marta
Published: July 2002

Covalent modification cycles are ubiquitous. Theoretical studies have suggested that they serve to increase sensitivity. However, this suggestion has not been corroborated experimentally
in vivo.
Here, we demonstrate that the assumptions of the theoretical studies, i.e., irreversibility and absence of product inhibition, were not trivial: when the conversion reactions are close to equilibrium or saturated by their product, “zero-order” ultrasensitivity disappears. For high sensitivities to arise, not only substrate saturation (zero-order) but also high equilibrium constants and low product saturation are required. Many covalent modification cycles are catalyzed by one bifunctional ‘ambiguous’ enzyme rather than by two independent proteins. This makes high substrate concentration and low product concentration for both reactions of the cycle inconsistent; such modification cycles cannot have high responses. Defining signal strength as ratios of modified (e.g., phosphorylated) over unmodified protein, signal-to-signal response sensitivity equals 1: signal strength should remain constant along a cascade of ambiguous modification cycles. We also show that the total concentration of a signalling effector protein cannot affect the signal emanating from a modification cycle catalyzed by an ambiguous enzyme if the ratio of the two forms of the effector protein is not altered. This finding may explain the experimental result that the pivotal signal transduction protein PII plus its paralogue GlnK do not control steady-state N-signal transduction in
Escherichia coli.
It also rationalizes the absence of strong phenotypes for many signal-transduction proteins. Emphasis on extent of modification of these proteins is perhaps more urgent than transcriptome analysis.
The role of intersection topography in bond selectivity ofcis-transphotoisomerization
Authors: Ben-Nun M. , Molnar F. , Schulten K. , Martínez Todd J.
Published: July 2002

Ab initiomethods are used to characterize the ground and first excited state of the chromophore in the rhodopsin family of proteins: retinal protonated Schiff base. Retinal protonated Schiff base has five double bonds capable of undergoing isomerization. Upon absorption of light, the chromophore isomerizes and the character of the photoproducts (e.g., 13-cis and 11-cis) depends on the environment, protein vs. solution. Ourab initiocalculations show that, in the absence of any specific interactions with the environment (e.g., discrete ordered charges in a protein), energetic considerations cannot explain the observed bond selectivity. We instead attribute the origin of bond selectivity to the shape (topography) of the potential energy surfaces in the vicinity of points of true degeneracy (conical intersections) between the ground and first excited electronic states. This provides a molecular example where a competition between two distinct but nearly isoenergetic photochemical reaction pathways is resolved by a topographical difference between two conical intersections.
Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein
Authors: Rohdich Felix , Hecht Stefan , Gärtner Katrin , Adam Petra , Krieger Cornelia , Amslinger Sabine , Arigoni Duilio , Bacher Adelbert , Eisenreich Wolfgang
Published: July 2002

Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-
d
-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier
in vivo
experiments with recombinant
Escherichia coli
strains showed that exogenous 1-deoxy-
d
-xylulose can be converted into 1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the
xylB
and
ispCDEFG
genes. This article describes the transformation of exogenous [U-
13
C
5
]1-deoxy-
d
-xylulose into a 5:1 mixture of [U-
13
C
5
]isopentenyl diphosphate and [U-
13
C
5
]dimethylallyl diphosphate by an
E. coli
strain engineered for the expression of the
ispH (lytB)
gene in addition to recombinant
xylB
and
ispCDEFG
genes.
A method for optimizing potential-energy functions by a hierarchical design of the potential-energy landscape: Application to the UNRES force field
Authors: Liwo Adam , Arłukowicz Piotr , Czaplewski Cezary , Ołdziej Stanisław , Pillardy Jarosław , Scheraga Harold A.
Published: July 2002

A method for optimizing potential-energy functions of proteins is proposed. The method assumes a hierarchical structure of the energy landscape, which means that the energy decreases as the number of native-like elements in a structure increases, being lowest for structures from the native family and highest for structures with no native-like element. A
level
of the hierarchy is defined as a family of structures with the same number of native-like elements (or
degree of native likeness
). Optimization of a potential-energy function is aimed at achieving such a hierarchical structure of the energy landscape by forcing appropriate free-energy gaps between hierarchy levels to place their energies in ascending order. This procedure is different from methods developed thus far, in which the energy gap and/or the Z score between the native structure and
all
non-native structures are maximized, regardless of the degree of native likeness of the non-native structures. The advantage of this approach lies in reducing the number of structures with decreasing energy, which should ensure the searchability of the potential. The method was tested on two proteins, PDB ID codes
1FSD
and
1IGD
, with an off-lattice united-residue force field. For
1FSD
, the search of the conformational space with the use of the conformational space annealing method and the newly optimized potential-energy function found the native structure very quickly, as opposed to the potential-energy functions obtained by former optimization methods. After even incomplete optimization, the force field obtained by using
1IGD
located the native-like structures of two peptides,
1FSD
and betanova (a designed three-stranded β-sheet peptide), as the lowest-energy conformations, whereas for the 46-residue N-terminal fragment of staphylococcal protein A, the native-like conformation was the second-lowest-energy conformation and had an energy 2 kcal/mol above that of the lowest-energy structure.
In vivo
evidence for a cochlear amplifier in the hair-cell bundle of lizards
Authors: Manley Geoffrey A. , Kirk Des L. , Köppl Christine , Yates Graeme K.
Published: July 2002

Vertebrate sensory hair cells achieve high sensitivity and
frequency selectivity by adding self-generated mechanical energy to
low-level signals. This allows them to detect signals that are smaller
than thermal molecular motion and to achieve significant resonance
amplitudes and frequency selectivity despite the viscosity of the
surrounding fluid. In nonmammals, a great deal of
in
vitro
evidence indicates that the active process responsible
for this amplification is intimately associated with the hair cells'
transduction channels in the stereovillar bundle. Here, we provide
in vivo
evidence of hair-cell bundle involvement in
active processes. Electrical stimulation of the inner ear of a lizard
at frequencies typical for this hearing organ induced low-level
otoacoustic emissions that could be modulated by low-frequency sound.
The unique modulation pattern permitted the tracing of the active
process involved to the stereovillar bundles of the sensory hair cells.
This supports the notion that, in nonmammals, the cochlear amplifier in
the hair cells is driven by a bundle motor system.
Intrinsic flexibility and gating mechanism of the potassium channel KcsA
Authors: Shen Yufeng , Kong Yifei , Ma Jianpeng
Published: July 2002

The gating mechanism of the potassium channel KcsA was studied by normal mode analysis. The results provided an atomic description of the locations of the pivot points and the motional features of key structural elements in the gating process. Two pivot points were found in the motions of the inner TM2 helical bundle that directly modulate the size of the central channel pore. One point is an intrasubunit hinge point that sharply divides the structural flexibility between the more rigid selectivity filter and the more mobile peripheral transmembrane helices. Such a division is vital for KcsA because it permits the large-scale motions of transmembrane helices required for the gating and, in the meantime, maintains the rigidity of the filter region essential for the selectivity. The other pivot point is an intersubunit one at which all four TM2 helices are bundled together. During the gating process, each TM2 helix undergoes a lever-like swinging motion pivoting on the intrasubunit hinge, and the entire TM2 bundle undergoes a concerted rotational motion around the central channel axis constrained around the intersubunit bundle point. This series of motions leads to a dramatic enlargement of the intracellular gate without loosening up the structural integrity.
Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles
Authors: Fernández César , Adeishvili Koba , Wüthrich Kurt
Published: July 2002

The
2
H,
13
C,
15
N-labeled,
148-residue integral membrane protein OmpX from
Escherichia
coli
was reconstituted with dihexanoyl phosphatidylcholine
(DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse
relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR
experiments and TROSY-type nuclear Overhauser enhancement spectra were
recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8
and 30°C. Complete sequence-specific NMR assignments for the
polypeptide backbone thus have been obtained. The
13
C
chemical shifts and the nuclear Overhauser effect data then resulted in
the identification of the regular secondary structure elements of
OmpX/DHPC in solution and in the collection of an input of
conformational constraints for the computation of the global fold of
the protein. The same type of polypeptide backbone fold is observed in
the presently determined solution structure and the previously reported
crystal structure of OmpX determined in the presence of the detergent
n
-octyltetraoxyethylene. Further structure refinement
will have to rely on the additional resonance assignment of partially
or fully protonated amino acid side chains, but the present data
already demonstrate that relaxation-optimized NMR techniques open novel
avenues for studies of structure and function of integral membrane
proteins.
NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response
Authors: Dweik Raed A. , Comhair Suzy A. A. , Gaston Benjamin , Thunnissen Frederik B. J. M. , Farver Carol , Thomassen Mary Jane , Kavuru Mani , Hammel Jeffrey , Abu-Soud Husam M. , Erzurum Serpil C.
Published: July 2002

A wealth of evidence supports increased NO (NO
⋅
) in asthma,
but its roles are unknown. To investigate how NO participates in
inflammatory airway events in asthma, we measured NO
⋅
and
NO
⋅
chemical reaction products [nitrite, nitrate,
S
-nitrosothiols (SNO), and nitrotyrosine] before,
immediately and 48 h after bronchoscopic antigen (Ag) challenge of
the peripheral airways in atopic asthmatic individuals and nonatopic
healthy controls. Strikingly, NO

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was the only
NO
⋅
derivative to increase during the immediate Ag-induced
asthmatic response and continued to increase over 2-fold at 48 h
after Ag challenge in contrast to controls [
P
<
0.05]. NO

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was not affected by Ag challenge at 10
min or 48 h after Ag challenge. Although SNO was not detectable in
asthmatic airways at baseline or immediately after Ag, SNO increased
during the late response to levels found in healthy controls. A model
of NO
⋅
dynamics derived from the current findings predicts that
NO
⋅
may have harmful effects through formation of peroxynitrite,
but also subserves an antioxidant role by consuming reactive oxygen
species during the immediate asthmatic response, whereas nitrosylation
during the late asthmatic response generates SNO, safe reservoirs for
removal of toxic NO
⋅
derivatives.
Genomic and genetic dissection of an archaeal regulon
Authors: Baliga Nitin S. , Kennedy Sean P. , Ng Wailap Victor , Hood Leroy , DasSarma Shiladitya
Published: July 2002

The extremely halophilic archaeon
Halobacterium
sp.
NRC-1 can grow phototrophically by means of light-driven proton pumping
by bacteriorhodopsin in the purple membrane. Here,
we show by genetic analysis of the wild type, and insertion and
double-frame shift mutants of Bat that this transcriptional regulator
coordinates synthesis of a structural protein and a chromophore for
purple membrane biogenesis in response to both light and oxygen.
Analysis of the complete
Halobacterium
sp. NRC-1 genome
sequence showed that the regulatory site, upstream activator sequence
(UAS), the putative binding site for Bat upstream of the bacterio-opsin
gene (
bop
), is also present upstream to the other
Bat-regulated genes. The transcription regulator Bat contains a
photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type
helix–turn–helix DNA binding motif. We also provide evidence for
involvement of the PAS/PAC domain of Bat in redox-sensing activity by
genetic analysis of a purple membrane overproducer. Five additional
Bat-like putative regulatory genes were found, which together are
likely to be responsible for orchestrating the complex response of this
archaeon to light and oxygen. Similarities of the
bop
-like UAS and transcription factors in diverse
organisms, including a plant and a γ-proteobacterium, suggest an
ancient origin for this regulon capable of coordinating light and
oxygen responses in the three major branches of the evolutionary tree
of life. Finally, sensitivity of four of five regulon genes to DNA
supercoiling is demonstrated and correlated to presence of alternating
purine–pyrimidine sequences (RY boxes) near the regulated promoters.
Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein
Authors: Kim Soo-Hyun M. , Azam Tania , Yoon Do-Young , Reznikov Leonid L. , Novick Daniela , Rubinstein Menachem , Dinarello Charles A.
Published: July 2002

IL-18 can be considered a proinflammatory cytokine mediating
disease as well as an immunostimulatory cytokine that is important for
host defense against infection and cancer. The high-affinity,
constitutively expressed, and circulating IL-18 binding protein
(IL-18BP), which competes with cell surface receptors for IL-18 and
neutralizes IL-18 activity, may act as a natural antiinflammatory as
well as immunosuppressive molecule. In the present studies, the IL-18
precursor caspase-1 cleavage site was changed to a factor Xa site, and,
after expression in
Escherichia coli
, mature IL-18 was
generated by factor Xa cleavage. Mature IL-18 generated by factor Xa
cleavage was fully active. Single point mutations in the mature IL-18
peptide were made, and the biological activities of the wild-type (WT)
IL-18 were compared with those of the mutants. Mutants E42A and K89A
exhibited 2-fold increased activity compared with WT IL-18. A double
mutant, E42A plus K89A, exhibited 4-fold greater activity.
Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus
K89A compared with WT IL-18. The K89A mutant was intermediate in being
neutralized by IL-18BP, whereas neutralization of the E42A mutant was
comparable to that in the WT IL-18. The identification of E42 and K89
in the mature IL-18 peptide is consistent with previous modeling
studies of IL-18 binding to IL-18BP and explains the unusually high
affinity of IL-18BP for IL-18.
The origin of atmospheric oxygen on Earth: The innovation of oxygenic photosynthesis
Authors: Dismukes G. C. , Klimov V. V. , Baranov S. V. , Kozlov Yu. N. , DasGupta J. , Tyryshkin A.
Published: July 2002

The evolution of O2-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO2) is a more efficient alternative substrate than water for O2production by oxygenic phototrophs. This analysis clarifies the origin of the long debated “bicarbonate effect” on photosynthetic O2production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O2evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.
Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: Differential binding of P and M helical enantiomers
Authors: Meistermann Isabelle , Moreno Virtudes , Prieto Maria J. , Moldrheim Erlend , Sletten Einar , Khalid Syma , Rodger P. Mark , Peberdy Jemma C. , Isaac Christian J. , Rodger Alison , Hannon Michael J.
Published: July 2002

We have designed a synthetic tetracationic metallo-supramolecular cylinder that targets the major groove of DNA with a binding constant in excess of 10
7
M
−1
and induces DNA bending and intramolecular coiling. The two enantiomers of the helical molecule bind differently to DNA and have different structural effects. We report the characterization of the interactions by a range of biophysical techniques. The M helical cylinder binds to the major groove and induces dramatic intramolecular coiling. The DNA bending is less dramatic for the P enantiomer.
Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments
Authors: Nakano Koichi , Chen Jing , Tarr George E. , Yoshida Tatsuro , Flynn Julia M. , Bitensky Mark W.
Published: July 2002

Phosducin (Pd), a small protein found abundantly in photoreceptors,
is widely assumed to regulate light sensitivity in the rod outer
segment through interaction with the heterotrimeric G protein
transducin. But, based on histochemistry and Western blot analysis, Pd
is found almost entirely in the inner segment in both light and dark,
most abundantly near the rod synapse. We report a second small protein,
14-3-3, in the rod with a similar distribution. By immunoprecipitation,
phospho-Pd is found to interact with 14-3-3 in material from
dark-adapted retina, and this interaction is markedly diminished by
light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds
to inner segment G protein(s) in the light. From these results and
reported functions of 14-3-3, we have constructed a hypothesis for the
regulation of light sensitivity at the level of rod synapse. By
dissociating the Pd/14-3-3 complex, light enables both proteins to
function in this role.
Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells
Authors: MacAry Paul A. , Lindsay Margaret , Scott Mike A. , Craig Jenny I. O. , Luzio J. Paul , Lehner Paul J.
Published: July 2002

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.
Amyloid aggregates of the HET-s prion protein are infectious
Authors: Maddelein Marie-Lise , Dos Reis Suzana , Duvezin-Caubet Stéphane , Coulary-Salin Bénédicte , Saupe Sven J.
Published: July 2002

The [Het-s] infectious element of the filamentous fungus
Podospora anserina
is a prion. We have recently reported that recombinant HET-s protein aggregates
in vitro
into amyloid fibers.
In vivo
, the protein aggregates specifically in the [Het-s] prion strains. Here, we show that biolistic introduction of aggregated recombinant HET-s protein into fungal cells induces emergence of the [Het-s] prion with a high frequency. Thus, we demonstrate that prion infectivity can be created
de novo
,
in vitro
from recombinant protein in this system. Although the amyloid filaments formed from HET-s could transmit [Het-s] efficiently, neither the soluble form of the protein nor amorphous aggregates would do so. In addition, we have found that (
i
) [Het-s] infectivity correlates with the ability to convert HET-s to amyloids
in vitro
, (
ii
) [Het-s] infectivity is resistant to proteinase K digestion, and (
iii
) HET-s aggregates formed
in vivo
in [Het-s] strains have the ability to convert the recombinant protein to aggregates. Together, our data designate the HET-s amyloids as the molecular basis of [Het-s] prion propagation.
Simulations of β-hairpin folding confined to spherical pores using distributed computing
Authors: Klimov D. K. , Newfield D. , Thirumalai D.
Published: July 2002

We report the thermodynamics and kinetics of an off-lattice Go model β-hairpin from Ig-binding protein confined to an inert spherical pore. Confinement enhances the stability of the hairpin due to the decrease in the entropy of the unfolded state. Compared with their values in the bulk, the rates of hairpin formationincreasein the spherical pore. Surprisingly, the dependence of the rates on the pore radius,Rs, is nonmonotonic. The rates reach a maximum atRs/R\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{g,{\mathit{N}}}^{b}}}\end{equation*}\end{document}≃ 1.5, whereR\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{g,{\mathit{N}}}^{b}}}\end{equation*}\end{document}is the radius of gyration of the folded β-hairpin in the bulk. The denatured state ensemble of the encapsulated β-hairpin ishighly structuredeven at substantially elevated temperatures. Remarkably, a profound effect of confinement is evident even when the β-hairpin occupies less than a 10th of the sphere volume. Our calculations show that the emergence of substantial structure in the denatured state of proteins in inert pores is a consequence of confinement. In contrast, the structure of the bulk denatured state ensemble depends dramatically on the extent of denaturation.
Ca
2+
-binding activity of a COOH-terminal fragment of the
Drosophila
BK channel involved in Ca
2+
-dependent activation
Authors: Bian Shumin , Favre Isabelle , Moczydlowski Edward
Published: July 2002

Mutational and biophysical analysis suggests that an intracellular
COOH-terminal domain of the large conductance
Ca
2+
-activated K
+
channel (BK channel) contains
Ca
2+
-binding site(s) that are allosterically coupled to
channel opening. However the structural basis of Ca
2+
binding to BK channels is unknown. To pursue this question, we
overexpressed the COOH-terminal 280 residues of the
Drosophila
slowpoke
BK channel (Dslo-C280) as a FLAG- and
His
6
-tagged protein in
Escherichia coli
. We
purified Dslo-C280 in soluble form and used a
45
Ca
2+
-overlay protein blot assay to detect
Ca
2+
binding. Dslo-C280 exhibits specific binding of
45
Ca
2+
in comparison with various control
proteins and known EF-hand Ca
2+
-binding proteins. A
mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of
the “Ca-bowl” motif are changed to Asn, reduces
45
Ca
2+
-binding activity by 56%. By
electrophysiological assay, the corresponding D5N5 mutant of the
Drosophila
BK channel expressed in HEK293 cells exhibits
lower Ca
2+
sensitivity for activation and a shift of ≈+80
mV in the midpoint voltage for activation. This effect is associated
with a decrease in the Hill coefficient (N) for activation by
Ca
2+
and a reduction in apparent Ca
2+
affinity,
suggesting the loss of one Ca
2+
-binding site per monomer.
These results demonstrate a functional correlation between
Ca
2+
binding to a specific region of the BK protein and
Ca
2+
-dependent activation, thus providing a biochemical
approach to study this process.
Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography
Authors: Okada Tetsuji , Fujiyoshi Yoshinori , Silow Maria , Navarro Javier , Landau Ehud M. , Shichida Yoshinori
Published: July 2002

Activation of G protein-coupled receptors (GPCRs) is triggered and regulated by structural rearrangement of the transmembrane heptahelical bundle containing a number of highly conserved residues. In rhodopsin, a prototypical GPCR, the helical bundle accommodates an intrinsic inverse-agonist 11-
cis
-retinal, which undergoes photo-isomerization to the
all-trans
form upon light absorption. Such a trigger by the chromophore corresponds to binding of a diffusible ligand to other GPCRs. Here we have explored the functional role of water molecules in the transmembrane region of bovine rhodopsin by using x-ray diffraction to 2.6 Å. The structural model suggests that water molecules, which were observed in the vicinity of highly conserved residues and in the retinal pocket, regulate the activity of rhodopsin-like GPCRs and spectral tuning in visual pigments, respectively. To confirm the physiological relevance of the structural findings, we conducted single-crystal microspectrophotometry on rhodopsin packed in our three-dimensional crystals and show that its spectroscopic properties are similar to those previously found by using bovine rhodopsin in suspension or membrane environment.
High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry
Authors: Young Malin M. , Tang Ning , Hempel Judith C. , Oshiro Connie M. , Taylor Eric W. , Kuntz Irwin D. , Gibson Bradford W. , Dollinger Gavin
Published: July 2002

We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS
3
). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the β-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1β, to build an FGF-2 homology model with a backbone error of 4.8 Å (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation.
SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis
Authors: Chamberlain Luke H. , Burgoyne Robert D. , Gould Gwyn W.
Published: July 2002

Lipid rafts are microdomains present within membranes of most cell
types. These membrane microdomains, which are enriched in cholesterol
and glycosphingolipids, have been implicated in the regulation of
certain signal transduction and membrane traffic pathways. To
investigate the possibility that lipid rafts organize exocytotic
pathways in neuroendocrine cells, we examined the association of
proteins of the exocytotic machinery with rafts purified from PC12
cells. The target soluble
N-
ethylmaleimide-sensitive
factor attachment protein receptor (tSNARE) proteins syntaxin 1A and
synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to
be highly enriched in lipid rafts (≈25-fold). The vesicle SNARE
vesicle-associated membrane protein (VAMP)2 was also present in raft
fractions, but the extent of this recovery was variable. However,
further analysis revealed that the majority of VAMP2 was associated
with a distinct class of raft with different detergent solubility
characteristics to the rafts containing syntaxin 1A and SNAP-25.
Interestingly, no other studied secretory proteins were significantly
associated with lipid rafts, including SNARE effector proteins such as
nSec1. Chemical crosslinking experiments showed that
syntaxin1A/SNAP-25 heterodimers were equally present in raft and
nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected
only in nonraft fractions. SDS-resistance assays revealed that
raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact
with VAMP2. Finally, reduction of cellular cholesterol levels decreased
the extent of regulated exocytosis of dopamine from PC12 cells. The
results described suggest that the interaction of SNARE proteins with
lipid rafts is important for exocytosis and may allow structural and
spatial organization of the secretory machinery.
Siderophore-mediated signaling regulates virulence factor production in
Pseudomonas
aeruginosa
Authors: Lamont Iain L. , Beare Paul A. , Ochsner Urs , Vasil Adriana I. , Vasil Michael L.
Published: July 2002

Numerous bacteria secrete low molecular weight compounds termed siderophores that have a high affinity for iron ions. Siderophores have a well-documented role as iron-scavenging chemicals, chelating iron ions in the environment whereupon the ferrisiderophores reenter the bacterial cells by means of specific cell-surface receptors. The iron is then released for incorporation into bacterial proteins. Here we show that in addition to its role as an iron-scavenger, the siderophore pyoverdine that is secreted by
Pseudomonas aeruginosa
regulates the production of at least three virulence factors (exotoxin A, an endoprotease, and pyoverdine itself), which are major contributors to the ability of this bacterium to cause disease. Regulation occurs through a transmembrane signaling system that includes an outer membrane receptor for ferripyoverdine, a signal-transducing protein that is predicted to extend from the periplasm into the cytoplasm, and a sigma factor. Expression of genes that form part of the regulon is triggered by pyoverdine so that this siderophore acts as a signaling molecule to control the production of secreted products. Recognition that a siderophore acts as a signaling molecule has important implications for the understanding of interactions between bacterial cells.
A family of RRM-type RNA-binding proteins specific to plant mitochondria
Authors: Vermel Matthieu , Guermann Benoit , Delage Ludovic , Grienenberger Jean-Michel , Maréchal-Drouard Laurence , Gualberto José M.
Published: July 2002

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed bothin vitroand invivoand by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of theArabidopsis thalianamt RNA-binding protein genes is induced by low temperatures.
Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching
Authors: Williams Mark C. , Rouzina Ioulia , Wenner Jay R. , Gorelick Robert J. , Musier-Forsyth Karin , Bloomfield Victor A.
Published: July 2002

The nucleocapsid protein (NC) of HIV type 1 is a nucleic acid
chaperone that facilitates the rearrangement of nucleic acids into
conformations containing the maximum number of complementary base
pairs. We use an optical tweezers instrument to stretch single DNA
molecules from the helix to coil state at room temperature in the
presence of NC and a mutant form (SSHS NC) that lacks the two zinc
finger structures present in NC. Although both NC and SSHS NC
facilitate annealing of complementary strands through electrostatic
attraction, only NC destabilizes the helical form of DNA and reduces
the cooperativity of the helix-coil transition. In particular, we find
that the helix-coil transition free energy at room temperature is
significantly reduced in the presence of NC. Thus, upon NC binding, it
is likely that thermodynamic fluctuations cause continuous melting and
reannealing of base pairs so that DNA strands are able to rapidly
sample configurations to find the lowest energy state. The reduced
cooperativity allows these fluctuations to occur in the middle of
complex double-stranded structures. The reduced stability and
cooperativity, coupled with the electrostatic attraction generated by
the high charge density of NC, is responsible for the nucleic acid
chaperone activity of this protein.
ERK2 enters the nucleus by a carrier-independent mechanism
Authors: Whitehurst Angelique W. , Wilsbacher Julie L. , You Youngjai , Luby-Phelps Kate , Moore Mary Shannon , Cobb Melanie H.
Published: July 2002

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an
in vitro
import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.
Basic FGF mediates an immediate response of articular cartilage to mechanical injury
Authors: Vincent Tonia , Hermansson Monika , Bolton Mark , Wait Robin , Saklatvala Jeremy
Published: July 2002

The extracellularly regulated kinase (ERK), one of the three types of mitogen-activated kinases, was rapidly activated after cutting porcine articular cartilage either when maintained as explants or
in situ
. Cutting released a soluble ERK-activating factor from the cartilage, which was purified and identified by MS as basic fibroblast growth factor (bFGF). Experiments with neutralizing Abs to bFGF and an FGFR1 tyrosine kinase inhibitor showed that this growth factor was the major ERK-activating factor released after injury. Treating cartilage with the heparin-degrading enzyme heparitinase also caused release of bFGF, suggesting the presence of an extracellular store that is sequestered in the matrix and released upon damage. Basic FGF induced the synthesis of a number of chondrocyte proteins including matrix metalloproteinases 1 and 3, tissue inhibitor of metalloproteinases-1, and glycoprotein 38, which were identified by MS. The strong induction of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 suggests that bFGF could have a role in remodeling damaged tissue.
Amino acid transport System A resembles System N in sequence but differs in mechanism
Authors: Reimer Richard J. , Chaudhry Farrukh A. , Gray Andrew T. , Edwards Robert H.
Published: July 2002

Classical amino acid transport System A accounts for most of the Na
+
-dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short- and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling.
Regulation of CFTR Cl
−
channel gating by ATP binding and hydrolysis
Authors: Ikuma Mutsuhiro , Welsh Michael J.
Published: July 2002

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl
−
channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg
2+
prevents hydrolysis. We found that even in the absence of Mg
2+
, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg
2+
. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg
2+
further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg
2+
, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel.
Simple mechanochemistry describes the dynamics of kinesin molecules
Authors: Fisher Michael E. , Kolomeisky Anatoly B.
Published: July 2002

Recently, Block and coworkers [Visscher, K., Schnitzer, M. J., & Block, S. M. (1999)
Nature (London)
400, 184–189 and Schnitzer, M. J., Visscher, K. & Block, S. M. (2000)
Nat. Cell Biol.
2, 718–723] have reported extensive observations of individual kinesin molecules moving along microtubules
in vitro
under controlled loads,
F
= 1 to 8 pN, with [ATP] = 1 μM to 2 mM. Their measurements of velocity,
V
, randomness,
r
, stalling force, and mean run length,
L
, reveal a need for improved theoretical understanding. We show, presenting explicit formulae that provide a quantitative basis for comparing distinct molecular motors, that their data are satisfactorily described by simple, discrete-state, sequential stochastic models. The simplest (
N
= 2)-state model with fixed load-distribution factors and kinetic rate constants concordant with stopped-flow experiments, accounts for the global (
V
,
F
,
L
, [ATP]) interdependence and, further, matches relative acceleration observed under
assisting
loads. The randomness,
r
(
F
,[ATP]), is accounted for by a waiting-time distribution, ψ

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(
t
), [for the transition(s) following ATP binding] with a width parameter ν ≡
〈t〉
2
/〈(Δ
t
)
2
〉≃2.5, indicative of a dispersive stroke of mechanicity ≃0.6
or
of a few (≳ν − 1) further, kinetically coupled states: indeed,
N
= 4 (but not
N
= 3) models do well. The analysis reveals: (
i
) a substep of
d
0
= 1.8–2.1 nm on ATP binding (consistent with structurally based suggestions); (
ii
) comparable load dependence for ATP binding and unbinding; (
iii
) a strong load dependence for reverse hydrolysis and subsequent reverse rates; and (
iv
) a large (≳50-fold) increase in detachment rate, with a marked load dependence, following ATP binding.
Force and kinetic barriers to unzipping of the DNA double helix
Authors: Cocco Simona , Monasson Rémi , Marko John F.
Published: July 2002

A theory of the unzipping of double-stranded DNA is presented and is compared to recent micromanipulation experiments. It is shown that the interactions that stabilize the double helix and the elastic rigidity of single strands simply determine the sequence-dependent ≈12-pN force threshold for DNA strand separation. Using a semimicroscopic model of the binding between nucleotide strands, we show that the greater rigidity of the strands when formed into double-stranded DNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. The effects of this barrier are derived analytically. The force to keep the extremities of the molecule at a fixed distance, the kinetic rates for strand unpairing at fixed applied force, and the rupture force as a function of loading rate are calculated. The dependence of the kinetics and of the rupture force on molecule length is also analyzed.
Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: A structural clue to amyloid assembly
Authors: Otzen Daniel E. , Kristensen Ole , Oliveberg Mikael
Published: July 2002

Limited solubility and precipitation of amyloidogenic sequences such as the Alzheimer peptide (β-AP) are major obstacles to a molecular understanding of protein fibrillation and deposition processes. Here we have circumvented the solubility problem by stepwise engineering a β-AP homology into a soluble scaffold, the monomeric protein S6. The S6 construct with the highest β-AP homology crystallizes as a tetramer that is linked by the β-AP residues forming intermolecular antiparallel β-sheets. This construct also shows increased coil aggregation during refolding, and a 14-mer peptide encompassing the engineered sequence forms fibrils. Mutational analysis shows that intermolecular association is linked to the overall hydrophobicity of the sticky sequence and implies the existence of “structural gatekeepers” in the wild-type protein, that is, charged side chains that prevent aggregation by interrupting contiguous stretches of hydrophobic residues in the primary sequence.
Propagating conformational changes over long (and short) distances in proteins
Authors: Yu Edward W. , Koshland Daniel E.
Published: July 2002

The problem of the propagation of conformational changes over long
distances or through a closely packed protein is shown to fit a model
of a ligand-induced conformational change between two protein states
selected by evolution. Moreover, the kinetics of the pathway between
these states is also selected so that the energy of ligand binding and
the speed of the transition between conformational states are
physiologically appropriate. The crystallographic data of a wild-type
aspartate receptor that has negative cooperativity and a mutant that
has no cooperativity but has native transmembrane signaling are shown
to support this model.
A retroviral RNA kissing complex containing only two G⋅C base pairs
Authors: Kim Chul-Hyun , Tinoco Ignacio
Published: July 2002

The dimerization of viral RNA through noncovalent interactions at their 5′ ends is a key step in the life cycle of retroviruses. In Moloney murine leukemia virus, three stem-loops are important in this process. One is a self-complementary tetraloop (H1), but the other two stem-loops (H2, H3) contain highly conserved GACG tetraloops that are not self-complementary sequences. Using two-dimensional NMR, we determined the structure of the H3 stem-loop. Surprisingly, it forms a stable, homodimeric kissing complex through only two intermolecular G⋅C base pairs. Cross-strand interactions of the adenines adjacent to the intermolecular G⋅C base pairs, plus unusual strong electrostatic interactions around the base pairs, contribute to the unexpected stability. This structure shows how even stem-loops without self-complementary sequences can facilitate the intermolecular recognition between two identical RNAs, and thus initiate dimerization and encapsidation of retroviral RNAs.