bluuebunny/crossref_metadata_2025 huggingface dataset:
Filopodia Formation in the Absence of Functional WAVE- and Arp2/3-Complexes
Authors: Steffen Anika , Faix Jan , Resch Guenter P. , Linkner Joern , Wehland Juergen , Small J. Victor , Rottner Klemens , Stradal Theresia E.B.
Published: April 2006

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.
Fibronectin Rigidity Response through Fyn and p130Cas Recruitment to the Leading Edge
Authors: Kostic Ana , Sheetz Michael P.
Published: April 2006

Cell motility on extracellular matrices critically depends on matrix rigidity, which affects cell adhesion and formation of focal contacts. Receptor-like protein tyrosine phosphatase alpha (RPTPα) and the αvβ3integrin form a rigidity-responsive complex at the leading edge. Here we show that the rigidity response through increased spreading and growth correlates with leading edge recruitment of Fyn, but not endogenous c-Src. Recruitment of Fyn requires the palmitoylation site near the N-terminus and addition of that site to c-Src enables it to support a rigidity response. In all cases, the rigidity response correlates with the recruitment of the Src family kinase to early adhesions. The stretch-activated substrate of Fyn and c-Src, p130Cas, is also required for a rigidity response and it is phosphorylated at the leading edge in a Fyn-dependent process. A possible mechanism for the fibronectin rigidity response involves force-dependent Fyn phosphorylation of p130Cas with rigidity-dependent displacement. With the greater displacement of Fyn from p130Cas on softer surfaces, there will be less phosphorylation. These studies emphasize the importance of force and nanometer-level movements in cell growth and function.
In Vivo Dynamics of Rac-Membrane Interactions
Authors: Moissoglu Konstadinos , Slepchenko Boris M. , Meller Nahum , Horwitz Alan F. , Schwartz Martin A.
Published: April 2006

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (koff) of membrane-bound GFP-Rac. We find that koffis 0.048 s−1for wtRac and ∼10-fold less (0.004 s−1) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased kofffor wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on koff. These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.
GUP1ofSaccharomyces cerevisiaeEncodes anO-Acyltransferase Involved in Remodeling of the GPI Anchor
Authors: Bosson Régine , Jaquenoud Malika , Conzelmann Andreas
Published: April 2006

The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Δ cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Δ cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Δ phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.
Antimicrobial Activity of Indigofera suffruticosa
Authors: Leite Sônia Pereira , Vieira Jeymesson Raphael Cardoso , de Medeiros Paloma Lys , Leite Roberta Maria Pereira , de Menezes Lima Vera Lúcia , Xavier Haroudo Satiro , de Oliveira Lima Edeltrudes
Published: April 2006

Various organic and aqueous extracts of leaves of Indigofera suffruticosa Mill (Fabaceae) obtained by infusion and maceration were screened for their antibacterial and antifungal activities. The extracts were tested against 5 different species of human pathogenic bacteria and 17 fungal strains by the agar‐solid diffusion method. Most of the extracts were devoid of antifungal and antibacterial activities, except the aqueous extract of leaves of I. suffruticosa obtained by infusion, which showed strong inhibitory activity against the Gram‐positive bacteria Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 5000 µg ml−1. The MIC values to dermatophyte strains were 2500 µg ml−1 against Trichophyton rubrum (LM‐09, LM‐13) and Microsporum canis. This study suggests that aqueous extracts of leaves of I. suffruticosa obtained by infusion can be used in the treatment of skin diseases caused by dermatophytes.
Governance at Points of Corporate Transition: Networks and the Formation of the United Alkali Company, 1890–1895
Authors: Popp Andrew
Published: April 2006

This article examines the formation in 1890 of the United Alkali Company in order to explore new perspectives on the transition to a corporate economy. Its starting point is the process of transition itself, highlighting the role played by networks. This perspective promotes an emphasis on institutional environments and on patterns of relationships and behavior, as opposed to those of formal structure. Thus, the archives of the company are probed for evidence of how key agents conceived of the firm. Finally, governance systems are seen as interacting in a dynamic fashion with the institutional environment, and in particular with the balance of internal and external economies.
Sperm numbers vary between inter- and intra-population matings of the grasshopper
Chorthippus parallelus
Authors: Reinhardt Klaus
Published: April 2006

Comparing the reproductive output of intra- and inter-population matings is the most common way to assess whether post-mating reproductive isolation is caused by genetic incompatibilities. Such genetic incompatibility can however, only assume that the quantity of the post-mating signals involved does not differ between intra- and inter-population matings. This assumption may not be true because sexual selection predicts reduced mating effort towards low-quality mates and in many circumstances, allopatric partners are low-quality mates. Post-mating efforts may, therefore, be reduced in inter- compared to intra-population matings. Here, I test this crucial assumption by studying variation in one post-mating trait, sperm number, in crosses of two parapatric grasshopper populations. In both populations, males transferred fewer sperm to allopatric than sympatric females. If such plasticity with respect to population is common in other post-mating traits, differences between inter- and intra-population crosses may be more frequently caused by differences in sperm number rather than gamete incompatibility. Additionally, I found that sperm numbers declined less rapidly in the female storage organ of allopatric than sympatric females but its rate differed markedly between populations. This is discussed with respect to female adaptations to male traits.
Can predatory bird feathers be used as a non-destructive biomonitoring tool of organic pollutants?
Authors: Jaspers Veerle L.B , Voorspoels Stefan , Covaci Adrian , Eens Marcel
Published: April 2006

The monitoring of different types of pollutants that are released into the environment and that present risks for both humans and wildlife has become increasingly important. In this study, we examined whether feathers of predatory birds can be used as a non-destructive biomonitor of organic pollutants. We demonstrate that polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and polybrominated diphenyl ethers (PBDEs) are measurable in one single tail feather of common buzzards (
Buteo buteo
) and that levels in this feather and internal tissues are significantly related to each other (0.35<
r
<0.76 for all 43 buzzards; 0.46<
r
<0.84 when excluding 17 starved birds). Our findings provide the first indication that feathers of predatory birds could be useful in non-destructive biomonitoring of organic pollutants, although further validation may be necessary.
Crab scars reveal survival advantage of left-handed snails
Authors: Dietl Gregory P , Hendricks Jonathan R
Published: April 2006

Biological asymmetries are important elements of the structure and function of many living organisms. Using the Plio–Pleistocene fossil record of crab predation on morphologically similar pairs of right- and left-handed snail species, we show here for the first time, contrary to traditional wisdom, that rare left-handed coiling promotes survival from attacks by right-handed crabs. This frequency-dependent result influences the balance of selection processes that maintain left-handedness at the species level and parallels some social interactions in human cultures, such as sports that involve dual contests between opponents of opposite handedness.
On the equations governing the motion of an anisotropic poroelastic material
Authors: Altay Gülay , Dökmecİ M. Cengİz
Published: April 2006

We address Biot's equations governing the motion of an anisotropic fluid-saturated poroelastic material with certain properties. First, we investigate the uniqueness in solutions of the three-dimensional governing equations for the regular region of the poroelastic material and enumerate the conditions sufficient for the uniqueness. Next, by applying Hamilton's principle to the motion of the region, we obtain a variational principle that generates only the Biot–Newton equations and the associated natural boundary conditions. Then, by extending the variational principle for the region with an internal fixed surface of discontinuity through Legendre's transformation, we derive a six-field variational principle that operates on all the poroelastic field variables. The variational principle leads, as its Euler–Lagrange equations, to all the governing equations, including the jump conditions but the initial conditions, as a generalized version of the Hellinger–Reissner variational principle. Moreover, we consider the free vibrations of the region, and we discuss some basic properties of eigenvalues and present a variational formulation by Rayleigh's quotient. This work provides a standard tool with the features of variational principles when numerically solving the governing equations in heterogeneous media with finite element methods, treating the free vibrations and consistently deriving some one-dimensional/two-dimensional equations of the poroelastic region.
New summation relations for the Stieltjes constants
Authors: Coffey Mark W
Published: April 2006

The Stieltjes constants

have been of interest for over a century, yet their detailed behaviour remains under investigation. These constants appear in the Laurent expansion of the Hurwitz zeta function

about

. We obtain novel single and double summatory relations for

, including single summation relations for

and

, where
a
and
b
are real and
p
and
q
are positive integers. In addition, we obtain new integration formulae for the Hurwitz zeta function and a new expression for the Stieltjes constants

. Portions of the presentation show an intertwining of the theory of the hypergeometric function with that of the Hurwitz zeta function.
An invasive species induces rapid adaptive change in a native predator: cane toads and black snakes in Australia
Authors: Phillips Ben L , Shine Richard
Published: April 2006

Rapid environmental change due to human activities has increased rates of extinction, but some species may be able to adapt rapidly enough to deal with such changes. Our studies of feeding behaviour and physiological resistance to toxins reveal surprisingly rapid adaptive responses in Australian black snakes (Pseudechis porphyriacus) following the invasion of a lethally toxic prey item, the cane toad (Bufo marinus). Snakes from toad-exposed localities showed increased resistance to toad toxin and a decreased preference for toads as prey. Separate laboratory experiments suggest that these changes are not attributable to learning (we were unable to teach naive snakes to avoid toxic prey) or to acquired resistance (repeated sub-lethal doses did not enhance resistance). These results strongly suggest that black snake behaviour and physiology have evolved in response to the presence of toads, and have done so rapidly. Toads were brought to Australia in 1935, so these evolved responses have occurred in fewer than 23 snake generations.
Senescent birds redouble reproductive effort when ill: confirmation of the terminal investment hypothesis
Authors: Velando Alberto , Drummond Hugh , Torres Roxana
Published: April 2006

This study reports an experimental confirmation of the terminal investment hypothesis, a longstanding theoretical idea that animals should increase their reproductive effort as they age and their prospects for survival and reproduction decline. Previous correlational and experimental attempts to test this hypothesis have yielded contradictory results. In the blue-footed booby,Sula nebouxii, a long-lived bird, after initial increase, male reproductive success declines progressively with age. Before laying, males of two age classes were challenged with lipopolysaccharide to elicit an immune response, which induced symptoms of declining survival prospects. Reproductive success of immune-challenged mature males fell, while that of immune-challenged old males showed a 98% increase. These results demonstrate that senescent males with poor reproductive prospects increase their effort when those prospects are threatened, whereas younger males with good reproductive prospects do not.
Evolutionary history of a mosquito endosymbiont revealed through mitochondrial hitchhiking
Authors: Rasgon Jason L , Cornel Anthony J , Scott Thomas W
Published: April 2006

Due to cytoplasmic inheritance, spread of maternally inheritedWolbachiasymbionts can result in reduction of mitochondrial variation in populations. We examined sequence diversity of the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene inWolbachia-infected (South Africa (SA), California and Thailand) and uninfected (SA)Culex pipienscomplex populations. In total, we identified 12 haplotypes (A–L). In infected populations, 99% of individuals had haplotype K. In the uninfected SA population, 11 haplotypes were present, including K. Nuclear allozyme diversity was similar between infected and uninfected SA populations. Analysis of nuclear DNA sequences suggested that haplotype K presence in uninfected SACx. pipienswas probably due to a shared ancestral polymorphism rather than hybrid introgression. These data indicate thatWolbachiaspread has resulted in drastic reduction of mitochondrial variability in widely separatedCx. pipienscomplex populations. In contrast, the uninfected SA population is probably a cryptic species whereWolbachiaintrogression has been prevented by reproductive isolation, maintaining ancestral levels of mitochondrial diversity. Molecular clock analyses suggest that theWolbachiasweep occurred within the last 47 000 years. The effect ofWolbachiaon mitochondrial dynamics can provide insight on the potential forWolbachiato spread transgenes into mosquito populations to control vector-borne diseases.
Dinosaur biomechanics
Authors: Alexander R. McNeill
Published: April 2006

Biomechanics has made large contributions to dinosaur biology. It has enabled us to estimate both the speeds at which dinosaurs generally moved and the maximum speeds of which they may have been capable. It has told us about the range of postures they could have adopted, for locomotion and for feeding, and about the problems of blood circulation in sauropods with very long necks. It has made it possible to calculate the bite forces of predators such asTyrannosaurus, and the stresses they imposed on its skull; and to work out the remarkable chewing mechanism of hadrosaurs. It has shown us how some dinosaurs may have produced sounds. It has enabled us to estimate the effectiveness of weapons such as the tail spines ofStegosaurus. In recent years, techniques such as computational tomography and finite element analysis, and advances in computer modelling, have brought new opportunities. Biomechanists should, however, be especially cautious in their work on animals known only as fossils. The lack of living specimens and even soft tissues oblige us to make many assumptions. It is important to be aware of the often wide ranges of uncertainty that result.
A mathematical model of pacemaker activity recorded from mouse small intestine
Authors: Youm Jae Boum , Kim Nari , Han Jin , Kim Euiyong , Joo Hyun , Leem Chae Hun , Goto Gazunori , Noma Akinori , Earm Yung E
Published: April 2006

The pacemaker activity of interstitial cells of Cajal (ICCs) has been known to initiate the propagation of slow waves along the whole gastrointestinal tract through spontaneous and repetitive generation of action potentials. We studied the mechanism of the pacemaker activity of ICCs in the mouse small intestine and tested it using a mathematical model. The model includes ion channels, exchanger, pumps and intracellular machinery for Ca
2+
regulation. The model also incorporates inositol 1,4,5-triphosphate (IP
3
) production and IP
3
-mediated Ca
2+
release activities. Most of the parameters were obtained from the literature and were modified to fit the experimental results of ICCs from mouse small intestine. We were then able to compose a mathematical model that simulates the pacemaker activity of ICCs. The model generates pacemaker potentials regularly and repetitively as long as the simulation continues. The frequency was set at 20 min
−1
and the duration at 50% repolarization was 639 ms. The resting and overshoot potentials were −78 and +1.2 mV, respectively. The reconstructed pacemaker potentials closely matched those obtained from animal experiments. The model supports the idea that cyclic changes in [Ca
2+
]
i
and [IP
3
] play key roles in the generation of ICC pacemaker activity in the mouse small intestine.
Simulation of Ca
2+
-activated Cl
−
current of cardiomyocytes in rabbit pulmonary vein: implications of subsarcolemmal Ca
2+
dynamics
Authors: Leem Chae Hun , Kim Won Tae , Ha Jeong Mi , Lee Yoon Jin , Seong Hyeon Chan , Choe Han , Jang Yeon Jin , Youm Jae Boum , Earm Yung E
Published: April 2006

In recent studies, we recorded transiently activated outward currents by the application of three-step voltage pulses to induce a reverse mode of Na
+
–Ca
2+
exchange (NCX). We found that these currents were mediated by a Ca
2+
-activated Cl
−
current. Based on the recent reports describing the atrial Ca
2+
transients, the Ca
2+
transient at the subsarcolemmal space was initiated and then diffused into the cytosolic space. Because the myocardium in the pulmonary vein is an extension of the atrium, the Ca
2+
-activated Cl
−
current may reflect the subsarcolemmal Ca
2+
dynamics. We tried to predict the subsarcolemmal Ca
2+
dynamics by simulating these current traces. According to recent reports on the geometry of atrial myocytes, we assumed that there were three compartments of sarcoplasmic reticulum (SR): a network SR, a junctional SR and a central SR. Based on these structures, we also divided the cytosolic space into three compartments: the junctional, subsarcolemmal and cytosolic spaces. Geometry information and cellular capacitance suggested that there were essentially no T-tubules in these cells. The basic physical data, such as the compartmental volumes, the diffusion coefficients and the stability coefficients of the Ca
2+
buffers, were obtained from the literature. In the simulation, we incorporated the NCX, the L-type Ca
2+
channel, the rapid activating outward rectifier K
+
channel, the Na
+
–K
+
pump, the SR Ca
2+
-pump, the ryanodine receptor, the Ca
2+
-activated Cl
−
channel and the dynamics of Na
+
, K
+
, Ca
2+
and Cl
−
. In these conditions, we could successfully reconstruct the Ca
2+
-activated Cl
−
currents. The simulation allowed estimation of the Ca
2+
dynamics of each compartment and the distribution of the Ca
2+
-activated Cl
−
channel and the NCX in the sarcolemma on the junctional or subsarcolemmal space.
The role of heterogeneities and intercellular coupling in wave propagation in cardiac tissue
Authors: Steinberg Benjamin E , Glass Leon , Shrier Alvin , Bub Gil
Published: April 2006

Electrical heterogeneities play a role in the initiation of cardiac arrhythmias. In certain pathological conditions such as ischaemia, current sinks can develop in the diseased cardiac tissue. In this study, we investigate the effects of changing the amount of heterogeneity and intercellular coupling on wavefront stability in a cardiac cell culture system and a mathematical model of excitable media. In both systems, we observe three types of behaviour: plane wave propagation without breakup, plane wave breakup into spiral waves and plane wave block. In the theoretical model, we observe a linear decrease in propagation velocity as the number of heterogeneities is increased, followed by a rapid, nonlinear decrease to zero. The linear decrease results from the heterogeneities acting independently on the wavefront. A general scaling argument that considers the degree of system heterogeneity and the properties of the excitable medium is used to derive a dimensionless parameter that describes the interaction of the wavefront with the heterogeneities.
Modulating RssB activity: IraP, a novel regulator of σS stability in Escherichia coli
Authors: Bougdour Alexandre , Wickner Sue , Gottesman Susan
Published: April 2006

The σS subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. σS is highly unstable in exponentially growing cells, whereas its stability increases dramatically upon starvation or under certain stress conditions. The degradation of σS is controlled by the phosphorylatable adaptor protein RssB and the ClpXP protease. RssB specifically directs σS to ClpXP. An unanswered question is how RssB-mediated degradation of σS is blocked by conditions such as glucose or phosphate starvation. We report here the identification and characterization of a new regulator of σS stability, IraP (inhibitor of RssB activity during phosphate starvation), that stabilizes σS both in vivo and in vitro. Deletion of iraP interferes with σS stabilization during phosphate starvation, but not during carbon starvation, and has a partial effect in stationary phase and nitrogen starvation. IraP interferes with RssB-dependent degradation of σS through a direct protein–protein interaction with RssB. A point mutant of IraP was isolated and found to be defective both for inhibition of σS degradation and interaction with RssB. Our results reveal a novel mechanism of regulation of σS stability through the regulation of RssB activity and identify IraP as a member of a new class of regulators, the anti-adaptor proteins.
The role of Rat1 in coupling mRNA 3′-end processing to transcription termination: implications for a unified allosteric–torpedo model
Authors: Luo Weifei , Johnson Arlen W. , Bentley David L.
Published: April 2006

The torpedo model of transcription termination by RNA polymerase II proposes that a 5′–3′ RNA exonuclease enters at the poly(A) cleavage site, degrades the nascent RNA, and eventually displaces polymerase from the DNA. Cotranscriptional degradation of nascent RNA has not been directly demonstrated, however. Here we report that two exonucleases, Rat1 and Xrn1, both contribute to cotranscriptional degradation of nascent RNA, but this degradation is not sufficient to cause polymerase release. Unexpectedly, Rat1 functions in both 3′-end processing and termination by enhancing recruitment of 3′-end processing factors, including Pcf11 and Rna15. In addition, the cleavage factor Pcf11 reciprocally aids in recruitment of Rat1 to the elongation complex. Our results suggest a unified allosteric/torpedo model in which Rat1 is not a dedicated termination factor, but is an integrated component of the cleavage/polyadenylation apparatus.
Post-transcriptional small RNA pathways in plants: mechanisms and regulations
Authors: Vaucheret Hervé
Published: April 2006

Small RNAs are riboregulators that have critical roles in most eukaryotes. They repress gene expression by acting either on DNA to guide sequence elimination and chromatin remodeling, or on RNA to guide cleavage and translation repression. This review focuses on the various types of post-transcriptional small RNA-directed pathways in plants, describing their roles and their regulations.
Conformational changes of the glucocorticoid receptor ligand binding domain induced by ligand and cofactor binding, and the location of cofactor binding sites determined by hydrogen/deuterium exchange mass spectrometry
Authors: Frego Lee , Davidson Walter
Published: April 2006

HXMS (hydrogen/deuterium exchange mass spectrometry) of the glucocorticoid receptor ligand‐binding domain (GR LBD) complexed with the agonist dexamethasone and the antagonist RU‐486 is described. Variations in the rates of exchange were observed in regions consistent with the published crystal structures of GR LBD complexed with RU‐486 when compared with the GR dexamethasone complex. We also report the HXMS results for agonist‐bound GR LBD with the coactivator transcriptional intermediary factor 2 (TIF2) and anatagonist‐bound GR LBD with nuclear receptor corepressor (NCoR). Alterations in exchange rates observed for agonist‐bound GR LBD with TIF2 present were consistent with the published crystal structural contacts for the complex. Alterations in exchange rates observed for antagonist‐bound GR LBD with NCoR were a subset of those observed with TIF2 binding, suggesting a common or overlapping binding site for coactivator and corepressor.
New approaches to high‐throughput structure characterization of SH3 complexes: The example of Myosin‐3 and Myosin‐5 SH3 domains from S. cerevisiae
Authors: Musi Valeria , Birdsall Berry , Fernandez‐Ballester Gregorio , Guerrini Remo , Salvatori Severo , Serrano Luis , Pastore Annalisa
Published: April 2006

SH3 domains are small protein modules that are involved in protein–protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic‐based model system to investigate the structural rules dictating the SH3‐mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.
Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination
Authors: Columbus Linda , Lipfert Jan , Klock Heath , Millett Ian , Doniach Sebastian , Lesley Scott A.
Published: April 2006

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent‐solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein–detergent complexes were characterized with small angle X‐ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross‐linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent‐solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein–detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein–detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein–detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.
Inhibition of insulin fibrillogenesis with targeted peptides
Authors: Gibson Todd J. , Murphy Regina M.
Published: April 2006

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of ≥100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12–17 of full‐length insulin, was identified as a short peptide that interacts with full‐length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near‐equimolar concentrations. An effective binding domain and N‐terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two‐step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.
A comparative study of available software for high‐accuracy homology modeling: From sequence alignments to structural models
Authors: Nayeem Akbar , Sitkoff Doree , Krystek Stanley
Published: April 2006

An open question in protein homology modeling is, how well do current modeling packages satisfy the dual criteria of quality of results and practical ease of use? To address this question objectively, we examined homology‐built models of a variety of therapeutically relevant proteins. The sequence identities across these proteins range from 19% to 76%. A novel metric, the difference alignment index (DAI), is developed to aid in quantifying the quality of local sequence alignments. The DAI is also used to construct the relative sequence alignment (RSA), a new representation of global sequence alignment that facilitates comparison of sequence alignments from different methods. Comparisons of the sequence alignments in terms of the RSA and alignment methodologies are made to better understand the advantages and caveats of each method. All sequence alignments and corresponding 3D models are compared to their respective structure‐based alignments and crystal structures. A variety of protein modeling software was used. We find that at sequence identities >40%, all packages give similar (and satisfactory) results; at lower sequence identities (<25%), the sequence alignments generated by Profit and Prime, which incorporate structural information in their sequence alignment, stand out from the rest. Moreover, the model generated by Prime in this low sequence identity region is noted to be superior to the rest. Additionally, we note that DSModeler and MOE, which generate reasonable models for sequence identities >25%, are significantly more functional and easier to use when compared with the other structure‐building software.
Exploring protein interfaces with a general photochemical reagent
Authors: Gómez Gabriela E. , Cauerhff Ana , Craig Patricio O. , Goldbaum Fernando A. , Delfino José M.
Published: April 2006

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent‐accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. 3H‐DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein–protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG1 D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21, G117TDVQAWIR125, andG22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.
Solution structure of Asl1650, an acyl carrier protein from Anabaena sp. PCC 7120 with a variant phosphopantetheinylation‐site sequence
Authors: Johnson Margaret A. , Peti Wolfgang , Herrmann Torsten , Wilson Ian A. , Wüthrich Kurt
Published: April 2006

Cyanobacteria, such as Anabaena, produce a variety of bioactive natural products via polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), and hybrid peptide/polyketide pathways. The protein Asl1650, which is a member of the acyl carrier protein family from the cyanobacterium Anabaena sp. PCC 7120, is encoded in a region of the Anabaena genome that is rich in PKS and NRPS genes. To gain new insight into the physiological role of acyl carriers in Anabaena, the solution structure of Asl1650 has been solved by NMR spectroscopy. The protein adopts a twisted antiparallel four‐helix bundle fold, with a variant phosphopantetheine‐attachment motif positioned at the start of the second helix. Structure comparisons with proteins from other organisms suggest a likely physiological function as a discrete peptidyl carrier protein.
Rapid peptide‐based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C‐like protease by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Authors: Chu Ling‐Hon Matthew , Choy Wai‐Yan , Tsai Sau‐Na , Rao Zihe , Ngai Sai‐Ming
Published: April 2006

Severe acute respiratory syndrome coronavirus (SARS‐CoV) 3C‐like protease (3CLpro) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti‐SARS drug development. Here we present a rapid and high‐throughput screening method to study the substrate specificity of SARS‐CoV 3CLpro. Six target amino acid positions flanking the SARS‐CoV 3CLpro cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the “cartridge replacement” approach and was subjected to enzymatic cleavage by recombinant SARS‐CoV 3CLpro. Susceptibility of each peptide substrate to SARS‐CoV 3CLpro cleavage was monitored simultaneously by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS‐CoV 3CLpro‐specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS‐CoV 3CLpro that is facing the P1′ position is suggested to be occupied by acidic amino acids, thus the P1′ position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or β‐branching amino acids in P3 and P2′ positions may also hinder the binding of SARS‐CoV 3CLpro. This study generates a comprehensive overview of SARS‐CoV 3CLpro substrate specificity, which serves as the design basis of synthetic peptide‐based SARS‐CoV 3CLpro inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high‐throughput manner that is compatible for future automated analysis.
Affinity enhancement of an in vivo matured therapeutic antibody using structure‐based computational design
Authors: Clark Louis A. , Boriack‐Sjodin P. Ann , Eldredge John , Fitch Christopher , Friedman Bethany , Hanf Karl J.M. , Jarpe Matthew , Liparoto Stefano F. , Li You , Lugovskoy Alexey , Miller Stephan , Rushe Mia , Sherman Woody , Simon Kenneth , Van Vlijmen Herman
Published: April 2006

Improving the affinity of a high‐affinity protein–protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure‐based computational methods to optimize the binding affinity of an antibody fragment to the I‐domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd ∼7 nM) and the moderate resolution (2.8 Å) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high‐affinity quadruple mutant complex at 2.2 Å. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher‐affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single‐mutant success rate. Our results indicate that structure‐based computational design can be successfully applied to further improve the binding of high‐affinity antibodies.
Thermodynamics of unfolding of an integral membrane protein in mixed micelles
Authors: Sehgal Pankaj , Otzen Daniel E.
Published: April 2006

Quantitative studies of membrane protein folding and unfolding can be difficult because of difficulties with efficient refolding as well as a pronounced propensity to aggregate. However, mixed micelles, consisting of the anionic detergent sodium dodecyl sulfate and the nonionic detergent dodecyl malto‐side facilitate reversible and quantitative unfolding and refolding. The 4‐transmembrane helix protein DsbB from the inner membrane of Escherichia coli unfolds in mixed micelles according to a three‐state mechanism involving an unfolding intermediate I. The temperature dependence of the kinetics of this reaction between 15° and 45°C supports that unfolding from I to the denatured state D is accompanied by a significant decrease in heat capacity. For water‐soluble proteins, the heat capacity increases upon unfolding, and this is generally interpreted as the increased binding of water to the protein as it unfolds, exposing more surface area. The decrease in DsbB's heat capacity upon unfolding is confirmed by independent thermal scans. The decrease in heat capacity is not an artifact of the use of mixed micelles, since the water soluble protein S6 shows conventional heat‐capacity changes in detergent. We speculate that it reflects the binding of SDS to parts of DsbB that are solvent‐exposed in the native DM‐bound state. This implies that the periplasmic loops of DsbB are relatively unstructured. This anomalous thermodynamic behavior has not been observed for β‐barrel membrane proteins, probably because they do not bind SDS so extensively. Thus the thermodynamic behavior of membrane proteins appears to be intimately connected to their detergent‐binding properties.
The contribution of the residues from the main hydrophobic core of ribonuclease A to its pressure‐folding transition state
Authors: Font Josep , Benito Antoni , Lange Reinhard , Ribó Marc , Vilanova Maria
Published: April 2006

The role of hydrophobic interactions established by the residues that belong to the main hydrophobic core of ribonuclease A in its pressure‐folding transition state was investigated using the Φ‐value method. The folding kinetics was studied using pressure‐jump techniques both in the pressurization and depressurization directions. The ratio between the folding activation volume and the reaction volume (βp‐value), which is an index of the compactness or degree of solvation of the transition state, was calculated. All the positions analyzed presented fractional Φf‐values, and the lowest were those corresponding to the most critical positions for the ribonuclease A stability. The structure of the transition state of the hydrophobic core of ribonuclease A, from the point of view of formed interactions, is a relatively, uniformly expanded form of the folded structure with a mean Φf‐value of 0.43. This places it halfway between the folded and unfolded states. On the other hand, for the variants, the average of βp‐values is 0.4, suggesting a transition state that is 40% native‐like. Altogether the results suggest that the pressure‐folding transition state of ribonuclease A looks like a collapsed globule with some secondary structure and a weakened hydrophobic core. A good correlation was found between the Φf‐values and the Δβp‐values. Although the nature of the transition state inferred from pressure‐induced folding studies and the results of the protein engineering method have been reported to be consistent for other proteins, to the best of our knowledge this is the first direct comparison using a set of mutants.
The C‐terminal domain of the transcriptional corepressor CtBP is intrinsically unstructured
Authors: Nardini Marco , Svergun Dmitri , Konarev Peter V. , Spanò Stefania , Fasano Mauro , Bracco Chiara , Pesce Alessandra , Donadini Alessandra , Cericola Claudia , Secundo Francesco , Luini Alberto , Corda Daniela , Bolognesi Martino
Published: April 2006

C‐terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate‐binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide‐binding domain, involved in NAD(H)‐binding and dimerization. On the contrary, little is known about the structure of CtBP C‐terminal region (∼90 residues), hosting sites for post‐translational modifications. In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small‐angle X‐ray scattering, and we show that the CtBP C‐terminal region is intrinsically unstructured in the full‐length CtBP and in constructs lacking the substrate‐ and/or the nucleotide‐binding domains. The flexible nature of this protein region, and its structural transitions, may be instrumental for CtBP recognition and binding to diverse molecular partners.
ULTRASTRUCTURAL MORPHOLOGY OF MICROTHAMNION ZOOSPORES1
Authors: Watson Melvin William , Arnott Howard J.
Published: April 2006

The Ultrastructure of Microthamnion zoospores is described (exclusive of the finer details of the flagellar apparatus). The zoospores have a typical chlorophycean morphology but, in addition, many unique features. The chloroplasts contain starch but no pyrenoid. Thylakoids may run from one edge of the chloroplast to the other and usually anastomose into 2‐ to 8‐membered stacks. The internal morphology is highly polarized and characterized by an intimate proximity and constant spacing between many organelle membranes. All the organelles are asymmetrically distributed within the cell in a precise manner. The anterior region of the zoospore is attenuated into a neck which contains a single, massive mitochondrion. A fibrous rhizoplast lies beside the mitochondrion and appears to connect the flagellar apparatus directly with the outer membrane of the nuclear envelope. In addition, this outer membrane is extended over a distance of several microns to eventually lie in close proximity to the basal bodies. Oil vacuoles and lipid bodies are restricted to the posterior end of the zoospore.