bluuebunny/crossref_metadata_2025 huggingface dataset:
Sustained Activation of Cell Adhesion Is a Differentially Regulated Process in B Lymphopoiesis
Authors: Glodek Aleksandra M. , Honczarenko Marek , Le Yi , Campbell James J. , Silberstein Leslie E.
Published: February 2003

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell–derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage–specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.
A Mouse with a Loss-of-function Mutation in the c-Cbl TKB Domain Shows Perturbed Thymocyte Signaling without Enhancing the Activity of the ZAP-70 Tyrosine Kinase
Authors: Thien Christine B.F. , Scaife Robin M. , Papadimitriou John M. , Murphy Maria A. , Bowtell David D.L. , Langdon Wallace Y.
Published: February 2003

The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activated protein tyrosine kinases (PTKs); this targeting is considered essential for Cbl proteins to negatively regulate PTKs. Here, a loss-of-function mutation (G304E) in the c-Cbl TKB domain, first identified in Caenorhabditis elegans, was introduced into a mouse and its effects in thymocytes and T cells were studied. In marked contrast to the c-Cbl knockout mouse, we found no evidence of enhanced activity of the ZAP-70 PTK in thymocytes from the TKB domain mutant mouse. This finding contradicts the accepted mechanism of c-Cbl–mediated negative regulation, which requires TKB domain targeting of phosphotyrosine 292 in ZAP-70. However, the TKB domain mutant mouse does show aspects of enhanced signaling that parallel those of the c-Cbl knockout mouse, but these involve the constitutive activation of Rac and not enhanced PTK activity. Furthermore, the enhanced signaling in CD4+CD8+ double positive thymocytes appears to be compensated by the selective down-regulation of CD3 on mature thymocytes and peripheral T cells from both strains of mutant c-Cbl mice.
Regulatory T Cells Selectively Express Toll-like Receptors and Are Activated by Lipopolysaccharide
Authors: Caramalho Iris , Lopes-Carvalho Thiago , Ostler Dominique , Zelenay Santiago , Haury Matthias , Demengeot Jocelyne
Published: February 2003

Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens. Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products. We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function. We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RBlow CD25+) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8. Exposure of CD4+ CD25+ cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation. This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation. Most importantly, LPS treatment increases CD4+ CD25+ cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4+ CD45RBlow CD25− subset that when tested ex-vivo, scores negative. Moreover, LPS-activated Treg efficiently control naive CD4 T cell–dependent wasting disease. These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.
Transgenic Expression of the Activating Natural Killer Receptor Ly49H Confers Resistance to Cytomegalovirus in Genetically Susceptible Mice
Authors: Lee Seung-Hwan , Zafer Ahmed , de Repentigny Yves , Kothary Rashmi , Tremblay Michel L. , Gros Philippe , Duplay Pascale , Webb John R. , Vidal Silvia M.
Published: February 2003

Natural resistance to infection with mouse cytomegalovirus (MCMV) is controlled by a dominant locus, Cmv1. Cmv1 is linked to the Ly49 family of natural killer receptors on distal chromosome 6. While some studies localized Cmv1 as distal to the Ly49 gene cluster, genetic and functional analysis identified Ly49h as a pivotal factor in resistance to MCMV. The role of these two independent genomic domains in MCMV resistance was evaluated by functional complementation using transgenesis of bacterial artificial chromosomes (BAC) in genetically susceptible mice. Phenotypic and genetic characterization of the transgenic animals traced the resistance gene to a single region spanning the Ly49h gene. The appearance of the Ly49H protein in NK cells of transgenic mice coincided with the emergence of MCMV resistance, and there was a threshold Ly49H protein level associated with full recovery. Finally, transgenic expression of Ly49H in the context of either of the two independent susceptibility alleles, Cmv1sBALB or Cmv1sFVB, conferred resistance to MCMV infection. These results demonstrate that Ly49h is necessary and sufficient to confer MCMV resistance, and formally demonstrate allelism between Cmv1 and Ly49h. This panel of transgenic animals provides a unique resource to study possible pleiotropic effect of Cmv1.
In Vitro Evidence That Cytokine Receptor Signals Are Required for Differentiation of Double Positive Thymocytes into Functionally Mature CD8+ T Cells
Authors: Yu Qing , Erman Batu , Bhandoola Avinash , Sharrow Susan O. , Singer Alfred
Published: February 2003

CD4+8+ double positive (DP) thymocytes differentiate into CD4+ and CD8+ mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4−8+ thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8+ thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells.
Low Penetrance, Broad Resistance, and Favorable Outcome of Interleukin 12 Receptor β1 Deficiency
Authors: Fieschi Claire , Dupuis Stéphanie , Catherinot Emilie , Feinberg Jacqueline , Bustamante Jacinta , Breiman Adrien , Altare Frédéric , Baretto Richard , Le Deist Françoise , Kayal Samer , Koch Hartmut , Richter Darko , Brezina Martin , Aksu Guzide , Wood Phil , Al-Jumaah Suliman , Raspall Miquel , da Silva Duarte Alberto José , Tuerlinckx David , Virelizier Jean-Louis , Fischer Alain , Enright Andrea , Bernhöft Jutta , Cleary Aileen M. , Vermylen Christiane , Rodriguez-Gallego Carlos , Davies Graham , Blütters-Sawatzki Renate , Siegrist Claire-Anne , Ehlayel Mohammad S. , Novelli Vas , Haas Walther H. , Levy Jacob , Freihorst Joachim , Al-Hajjar Sami , Nadal David , de Moraes Vasconcelos Dewton , Jeppsson Olle , Kutukculer Necil , Frecerova Klara , Caragol Isabel , Lammas David , Kumararatne Dinakantha S. , Abel Laurent , Casanova Jean-Laurent
Published: February 2003

The clinical phenotype of interleukin 12 receptor β1 chain (IL-12Rβ1) deficiency and the function of human IL-12 in host defense remain largely unknown, due to the small number of patients reported. We now report 41 patients with complete IL-12Rβ1 deficiency from 17 countries. The only opportunistic infections observed, in 34 patients, were of childhood onset and caused by weakly virulent Salmonella or Mycobacteria (Bacille Calmette-Guérin -BCG- and environmental Mycobacteria). Three patients had clinical tuberculosis, one of whom also had salmonellosis. Unlike salmonellosis, mycobacterial infections did not recur. BCG inoculation and BCG disease were both effective against subsequent environmental mycobacteriosis, but not against salmonellosis. Excluding the probands, seven of the 12 affected siblings have remained free of case-definition opportunistic infection. Finally, only five deaths occurred in childhood, and the remaining 36 patients are alive and well. Thus, a diagnosis of IL-12Rβ1 deficiency should be considered in children with opportunistic mycobacteriosis or salmonellosis; healthy siblings of probands and selected cases of tuberculosis should also be investigated. The overall prognosis is good due to broad resistance to infection and the low penetrance and favorable outcome of infections. Unexpectedly, human IL-12 is redundant in protective immunity against most microorganisms other than Mycobacteria and Salmonella. Moreover, IL-12 is redundant for primary immunity to Mycobacteria and Salmonella in many individuals and for secondary immunity to Mycobacteria but not to Salmonella in most.
Interleukin 1 Receptor Dependence of Serum Transferred Arthritis Can be Circumvented by Toll-like Receptor 4 Signaling
Authors: Choe Jung-Yoon , Crain Brian , Wu Sarah R. , Corr Maripat
Published: February 2003

Inflammatory arthritis is associated with the release of a network of key cytokines. In T cell receptor transgenic K/BxN mice interleukin (IL)-1 plays a key role in joint swelling and destruction, as suggested by the ability of anti–IL-1receptor (IL-1R) antibody treatment to delay the onset and slow the progression of this disease. This mechanism is dependent on the signaling pathway intermediary myeloid differentiation factor 88 (MyD88), such that neither IL-1R nor MyD88-deficient mice developed visually detectable synovitis after transfer of arthritogenic sera. The Toll-like receptors (TLRs) share the same signaling pathway through MyD88 as the IL-1R. The administration of a TLR-4 ligand, lipopolysaccharide, concomitant with arthritogenic serum in IL-1 receptor–deficient mice resulted in acute paw swelling, but not in MyD88-deficient mice. Also, serum transferred arthritis was not sustained in TLR-4 mutant mice compared with controls. These results suggest that innate immune functions via TLR-4 might perpetuate inflammatory mechanisms and bypass the need for IL-1 in chronic joint inflammation.
Can transcriptome size be estimated from SAGE catalogs?
Authors: Stern Michael D. , Anisimov Sergey V. , Boheler Kenneth R.
Published: February 2003

Motivation: SAGE (Serial Analysis of Gene Expression) can be used to estimate the number of unique transcripts in a transcriptome. A simple estimator that corrects for sequencing and sampling errors was applied to a SAGE library (137 832 tags) obtained from mouse embryonic stem cells, and also to Monte Carlo simulated libraries generated using assumed distributions of‘ true’ expression levels consistent with the data.

Results: When the corrected data themselves were taken as the underlying model of ‘ground truth’, the estimator converged to the ‘ true’ value (53 535) only after counting 300 000 simulated tags, more than twice the number in the experiment. The SAGE data could also be well fit by a Monte Carlo model based on a truncated inverse-square distribution of expression levels, with 130 000 ‘true’ transcripts and 106 samples needed for convergence. We conclude that the size of a transcriptome is ill-determined from SAGE libraries of even moderately large size. In order to obtain a valid estimate, one must sample a number of tags inversely proportional to the lowest abundance level, which is not known a priori. This constrains the design of SAGE experiments intended to determine biological complexity.

Availability: The ‘homemade’ software used for this analysis was not designed for general or ‘production’ use, but the authors will be happy to share Fortran sourcecode with interested parties.

Contact: sternm@grc.nia.nih.gov

* To whom correspondence should be addressed.
Protein family annotation in a multiple alignment viewer
Authors: Johnson Jason M. , Mason Keith , Moallemi Ciamac , Xi Hualin , Somaroo Shyamal , Huang Enoch S.
Published: February 2003

Summary: The Pfaat protein family alignment annotation tool is a Java-based multiple sequence alignment editor and viewer designed for protein family analysis. The application merges display features such as dendrograms, secondary and tertiary protein structure with SRS retrieval, subgroup comparison, and extensive user-annotation capabilities.

Availability: The program and source code are freely available from the authors under the GNU General Public License at http://www.pfizerdtc.com

Contact: jason_johnson@merck.comenoch_huang@cambridge.pfizer.com

* To whom correspondence should be addressed.

† Present address: Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, WA 98034, USA.
MaxSubSeq: an algorithm for segment-length
optimization. The case study of the transmembrane
spanning segments
Authors: Fariselli Piero , Finelli Michele , Marchignoli Davide , Martelli Pier Luigi , Rossi Ivan , Casadio Rita
Published: February 2003

Motivation: A problem in predicting the topography of transmembrane proteins is the optimal localization of the transmembrane segments along the protein sequences, provided that each residue is associated with a propensity of being or not being included in the transmembrane protein region. From previous work it is known that post-processing of propensity signals with suited algorithms can greatly improve the quality and the accuracy of the predictions. In this paper we describe a general dynamic programming-like algorithm (MaxSubSeq, Maximal SubSequence) specifically designed to optimize the number and length of segments with constrained length in a given protein sequence. Previous application of our algorithm, has proved its effectiveness in the optimization task of both neural network and hidden Markov models output, and in this paper we present the detailed description of MaxSubSeq.

Results: We describe the application of MaxSubSeq to the location of both helical and beta strand transmembrane segments, optimizing the outputs derived with different predictive algorithms. For all-alpha transmembrane proteins we use both the standard Kyte–Doolittle (KD) hydropathy scale and the TMHMM predictor (http://www.cbs.dtu.dk/). Using a set of 188 well characterized membrane proteins, MaxSubSeq nearly doubles the correct location of transmembrane segments as compared to the standard KD hydrophobicity plot, reaching 51% accuracy. If MaxSubSeq is used to optimize the TMHMM method the accuracy increases from 68 to 72%. When used to regularize the prediction of beta transmembrane strands, obtained using both a neural network and a HMM based predictors, MaxSubSeq increases the accuracy per protein up to 72 and 73% respectively.

Availability: The program is available upon request to the authors, or it is accessible through our web server (http://gpcr.biocomp.unibo.it/predictors/)

Contact: piero@biocomp.unibo.itcasadio@alma.unibo.it

* To whom correspondence should be addressed.
PopHist: inferring population history from the spectrum of allele frequencies
Authors: Wooding Stephen
Published: February 2003

Summary: PopHist is a computer program that uses the frequency spectrum of alleles to: (a) estimate maximum likelihood parameters describing a population’s history; and (b) compare alternative hypotheses about population history using likelihood ratio tests. The program uses the matrix coalescent, a method for calculating theoretical frequency spectra that can be applied to sets of unlinked sites.

Availability: Source code and documentation are available at http://mombasa.anthro.utah.edu/wooding/PopHist

Contact: swooding@genetics.utah.edu
Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints
Authors: Marassi Francesca M. , Opella Stanley J.
Published: February 2003

A solid‐state NMR approach for simultaneous resonance assignment and three‐dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor “shotgun,” of 15N‐labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not. Shotgun NMR short‐circuits the laborious and time‐consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins. As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination. A total of five two‐dimensional 1H/15N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively 15N‐labeled samples, were sufficient to determine the structure of the membrane‐bound form of the 50‐residue major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three‐dimensional structure of the protein with atomic resolution. The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy.

[The structure we determined for the membrane‐bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.]
Hydrophobicity of transmembrane proteins: Spatially profiling the distribution
Authors: Silverman B. David
Published: February 2003

A hallmark of soluble globular protein tertiary structure is a hydrophobic core and a protein exterior populated predominantly by hydrophilic residues. Recent hydrophobic moment profiling of the spatial distribution of 30 globular proteins of diverse size and structure had revealed features of this distribution that were comparable. Analogous profiling of the hydrophobicity distribution of the α‐helical buried bundles of several transmembrane proteins, as the lipid/protein interface is approached from within the bilayer, reveals spatial hydrophobicity profiles that contrast with those obtained for the soluble proteins. The calculations, which enable relative changes of hydrophobicity to be simply identified over the entire spatial extent of the multimer within the lipid bilayer, show the accumulated zero‐order moments of the bundles to be mainly inverted with respect to that found for the soluble proteins. This indicates a statistical increase in the average residue hydrophobic content as the lipid bilayer is approached. This result differs from that of a relatively recent calculation and qualitatively agrees with earlier calculations involving lipid exposed and buried residues of the α‐helices of transmembrane proteins. Spatial profiling, over the entire spatial extent of the multimer with scaled values of residue hydrophobicity, provides information that is not available from calculations using lipid exposure alone.
Backbone dynamics of the human MIA protein studied by 15N NMR relaxation: Implications for extended interactions of SH3 domains
Authors: Stoll Raphael , Renner Christian , Buettner Reinhard , Voelter Wolfgang , Bosserhoff Anja‐Katrin , Holak Tad A.
Published: February 2003

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by 15N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting β‐sheets, such as the RT‐loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT‐loop, the n‐Src loop, for the loop comprising residues 13 to 19, which we refer to as the“disulfide loop”, in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte‐derived protein (FDP)/MIA‐like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain‐like fold in solution with expanded ligand interactions.
The hydration of amides in helices; a comprehensive picture from molecular dynamics, IR, and NMR
Authors: Walsh Scott T.R. , Cheng Richard P. , Wright Wayne W. , Alonso Darwin O.V. , Daggett Valerie , Vanderkooi Jane M. , DeGrado William F.
Published: February 2003

We examined the hydration of amides of α3D, a simple, designed three‐helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy (∼50%–70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I′ band) of a group of surface Ala residues. The surface‐exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I′ bands of the buried 13C‐labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen‐deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4–7 residues N‐terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 310/α/π‐helical conformation.
In vitro unfolding, refolding, and polymerization of human γD crystallin, a protein involved in cataract formation
Authors: Kosinski‐Collins Melissa S. , King Jonathan
Published: February 2003

Human γD crystallin (HγD‐Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174‐residue primarily β‐sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HγD‐Crys are implicated in early‐onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild‐type HγD‐Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37°C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild‐type HγD‐Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion‐tryptophan interactions. Aggregation of refolding intermediates of HγD‐Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HγD‐Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HγD‐Crys aggregation may provide clues to understanding age‐onset cataract formation in vivo.
Enhanced correction methods for hydrogen exchange‐mass spectrometric studies of amyloid fibrils
Authors: Kheterpal Indu , Wetzel Ronald , Cook Kelsey D.
Published: February 2003

We describe methods for minimization of and correction for artifactual forward and backward exchange occurring during hydrogen exchange–mass spectrometric (HX–MS) studies of amyloid fibrils of the Aβ(1–40) peptide. The quality of the corrected data obtained using published and new correction algorithms is evaluated quantitatively. Using the new correction methods, we have determined that 20.1 ± 1.4 of the 39 backbone amide hydrogens in Aβ(1–40) exchange with deuteriums in 100 h when amyloid fibrils of this peptide are suspended in D2O. These data reinforce our previous conclusions based on uncorrected data that amyloid fibrils contain a rigid protective core structure that involves only about half of the Aβ backbone amides. The methods developed here should be of general value for HX–MS studies of amyloid fibrils and other protein aggregates.
Mutations in the B1 domain of protein G that delay the onset of amyloid fibril formation in vitro
Authors: Ramírez‐Alvarado Marina , Cocco Melanie J. , Regan Lynne
Published: February 2003

We previously reported that under certain experimental conditions, many variants of the B1 domain of IgG‐binding protein G from Streptococcus form fibrils reproducibly. The variant I6T53 was the focus of the present study because the lag phase in the kinetics of fibril formation by this variant is significantly longer than that of other variants. This lag phase is distinguished by changes in both intrinsic fluorescence intensity and in light scattering of the protein. NMR diffusion measurements suggest that the soluble protein during the lag phase is monomeric. The kinetic profiles of fibril formation are found to depend on experimental conditions. The first kinetic phase diminishes almost completely when the reaction is seeded with preformed amyloid fibrils.
Solution structure of termicin, an antimicrobial peptide from the termite Pseudacanthotermes spiniger
Authors: Da Silva Pedro , Jouvensal Laurence , Lamberty Mireille , Bulet Philippe , Caille Anita , Vovelle Françoise
Published: February 2003

The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two‐dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine‐rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an α‐helical segment (Phe4–Gln14) and a two‐stranded (Phe19–Asp25 and Gln28–Phe33) antiparallel β‐sheet forming a “cysteine stabilized αβ motif” (CSαβ) also found in antibacterial and antifungal defensins from insects and from plants. Interestingly, termicin shares more structural similarities with the antibacterial insect defensins and with MGD‐1, a mussel defensin, than with the insect antifungal defensins such as drosomycin and heliomicin. These structural comparisons suggest that global fold alone does not explain the difference between antifungals and antibacterials. The antifungal properties of termicin may be related to its marked hydrophobicity and its amphipatic structure as compared to the antibacterial defensins. [SWISS‐PROT accession number: Termicin (P82321); PDB accession number: 1MM0.]
Prediction of β‐turns in proteins from multiple alignment using neural network
Authors: Kaur Harpreet , Raghava Gajendra Pal Singh
Published: February 2003

A neural network‐based method has been developed for the prediction of β‐turns in proteins by using multiple sequence alignment. Two feed‐forward back‐propagation networks with a single hidden layer are used where the first‐sequence structure network is trained with the multiple sequence alignment in the form of PSI‐BLAST–generated position‐specific scoring matrices. The initial predictions from the first network and PSIPRED‐predicted secondary structure are used as input to the second structure‐structure network to refine the predictions obtained from the first net. A significant improvement in prediction accuracy has been achieved by using evolutionary information contained in the multiple sequence alignment. The final network yields an overall prediction accuracy of 75.5% when tested by sevenfold cross‐validation on a set of 426 nonhomologous protein chains. The corresponding Qpred, Qobs, and Matthews correlation coefficient values are 49.8%, 72.3%, and 0.43, respectively, and are the best among all the previously published β‐turn prediction methods. The Web server BetaTPred2 (http://www.imtech.res.in/raghava/betatpred2/) has been developed based on this approach.
Reinvestigation of the proposed folding and self‐association of the Neuropeptide Head Activator
Authors: Lai Jonathan R. , Gellman Samuel H.
Published: February 2003

The Neuropeptide Head Activator (HA), pGlu‐Pro‐Pro‐Gly‐Gly‐Ser‐Lys‐Val‐Ile‐Leu‐Phe (pGlu is pyroglutamic acid), is involved in head‐specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher‐organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (Kd ≈ 1 nM). This dimerization was proposed to occur via antiparallel β‐sheet formation between the Lys7‐Phe11 segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by 1H‐NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys7‐Phe11 fragment to form antiparallel β‐sheet by designing peptides in which two such fragments are joined by a two‐residue linker. These peptides were intended to form stable β‐hairpin structures with cross‐strand interactions that mimic those of the proposed HA dimer interface. We found that the HA‐derived fragments may be induced to form intramolecular β‐sheet, albeit only weakly, when linked by the highly β‐hairpin‐promoting d‐Pro‐Gly turn, but not when linked by the more flexible Gly‐Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.
Purification of correctly oxidized MHC class I heavy‐chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding
Authors: Ferré Henrik , Ruffet Emmanuel , Blicher Thomas , Sylvester‐Hvid Christina , Nielsen Lise Lotte B. , Hobley Timothy J. , Thomas Owen R.T. , Buus Søren
Published: February 2003

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC‐I) heavy‐chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy‐chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1) correct folding can only take place in combined presence of β2‐microglobulin and a binding peptide; and (2) optimal in vitro conditions for disulfide bond formation (∼pH 8) and peptide binding (∼pH 6.6) are far from complementary. Here we present a two‐step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy‐chain molecules with correct disulfide bonding are formed under non‐reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond–assisted folding in the presence of β2‐microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.
WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains
Authors: Otte Livia , Wiedemann Urs , Schlegel Brigitte , Pires José Ricardo , Beyermann Michael , Schmieder Peter , Krause Gerd , Volkmer‐Engert Rudolf , Schneider‐Mergener Jens , Oschkinat Hartmut
Published: February 2003

WW domains mediate protein–protein interactions in a number of different cellular functions by recognizing proline‐containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty‐two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/ϕ)P(p,g)PPpR, (p/ϕ)PPRgpPp, PPLPp, (p/ξ)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group‐specific WW domain consensus sequences were identified. For PPxY‐recognizing domains, phospho‐tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)‐specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)‐recognition activity for WW domains, which were not assayed. PPx(Y/poY)‐binding was found to be a common property of NEDD4‐like ubiquitin ligases.
Protein folding coupled to DNA binding in the catalytic domain of bacteriophage λ integrase detected by mass spectrometry
Authors: Kamadurai Hari B. , Subramaniam Srisunder , Jones R. Benjamin , Green‐Church Kari B. , Foster Mark P.
Published: February 2003

Bacteriophage λ integrase (λ‐Int) is the prototypical member of a large family of enzymes that catalyze site‐specific DNA recombination via single‐strand cleavage and the formation of a Holliday junction intermediate. Crystallographic and biochemical evidence indicate that substantial conformational change (i.e., folding) in the catalytic domain of the protein is required for substrate recognition and catalysis. We have examined the solution conformation of the catalytic domain (C170) in the absence and presence of a cognate “half‐site” DNA oligonucleotide by electrospray ionization mass spectrometry, and circular dichroism and fluorescence spectroscopy. The distribution of ions in the positive ion electrospray mass spectrum of the free protein reveals the presence of three distinct species in solution, one corresponding to the folded protein, one to the unfolded protein, and one to a dimer. In the presence of DNA, ions are observed only for the protein–DNA complex and the folded form of the free protein. We therefore conclude that DNA binding stabilizes the global fold of the protein in a manner that is consistent with folding‐coupled target recognition as a mechanism to control site‐specific recombination. Furthermore, we find that inspection of the charge state distribution of ions in electrospray mass spectra provides a quick and effective means to identify conformational heterogeneity of proteins in solution and to investigate dynamic protein–nucleic acid interactions.
Crystal structure of a defective folding protein
Authors: Saul Frederick A. , Mourez Michaël , Vulliez‐le Normand Brigitte , Sassoon Nathalie , Bentley Graham A. , Betton Jean‐Michel
Published: February 2003

Maltose‐binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32→Asp and Ile 33→Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat‐shock periplasmic peptidyl‐prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose‐binding activity comparable with that of the wild‐type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose‐bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 Å resolution. The conformation of MalE31 closely resembles that of wild‐type MalE, with very small displacements of the mutated residues located in the loop connecting the first α‐helix to the first β‐strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild‐type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.
Equilibrium and kinetic studies on folding of canine milk lysozyme
Authors: Van Dael Herman , Haezebrouck Petra , Joniau Marcel
Published: February 2003

Thermal and chemical unfolding studies of the calcium‐binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped‐flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo‐canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s−1. Furthermore, the results show that, unlike its drastic effect on the stability, Ca2+‐binding only marginally affects the refolding kinetics. During the refolding process of apo‐CL non‐native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non‐native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.
A PDZ domain‐based assay for measuring HIV protease activity: Assay design considerations
Authors: Hamilton Aaron C. , Inglese James , Ferrer Marc
Published: February 2003

We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain•peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ‐binding motif is exposed, and the product sequence bound specifically by a Eu3+chelate‐labeled GST–PDZ ([Eu3+]GST–PDZ). The practical applicability of this PDZ‐based detection method is determined by the affinity of the PDZ domain•peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ‐based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD‐95 was used, which preferentially recognizes the consensus sequence Ser‐X‐Val‐COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser‐X‐Leu‐COOH, can be used to extend the flexibility in the recognition of the carboxy‐terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.
Competing intrachain interactions regulate the formation of β‐sheet fibrils in bovine PrP peptides
Authors: Tahiri‐Alaoui Abdessamad , Bouchard Mario , Zurdo Jesús , James William
Published: February 2003

At the heart of the pathogenesis of transmissible spongiform encephalopathies (TSEs), such as BSE, scrapie, and Creutzfeldt–Jakob disease, lies a poorly understood structural rearrangement of PrP, an abundant glycoprotein of the nervous and lymphoid systems. The normal form (PrPC), rich in α‐helix, converts into an aberrant β‐sheet‐dominated form (PrPSc), which seems to be at the center of the pathotoxic symptoms observed in TSEs. To understand this process better at a molecular level, we have studied the interactions between different peptides derived from bovine PrP and their structural significance. We show that two unstructured peptides derived from the central region of bovine PrP, residues 115–133 and 140–152, respectively, interact stoichiometrically under physiological conditions to generate β‐sheet‐dominated fibrils. However, when both peptides are incubated in the presence of a third peptide derived from an adjoining α‐helical region (residues 153–169), the formation of β‐sheet‐rich fibrils is abolished. These data indicate that native PrPC helix 1 might inhibit the strong intrinsic β‐sheet‐forming propensity of sequences immediately N‐terminal to the globular core of PrPC, by keeping in place intrachain interactions that would prevent these amyloidogenic regions from triggering aggregation. Moreover, these results indicate new ways in which PrPSc formation could be prevented.
The effect of the polyproline II (PPII) conformation on the denatured state entropy
Authors: Ferreon Josephine C. , Hilser Vincent J.
Published: February 2003

Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM‐5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard‐sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, ∼30% and ∼10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.
Microstructural and surface properties of cobalt containing amorphous carbon thin film deposited by a filtered cathodic vacuum arc
Authors: Chua Daniel H. C. , Milne W. I. , Tay B. K. , Zhang P. , Ding X. Z.
Published: February 2003

Cobalt containing tetrahedral amorphous carbon (ta-C:Co) films were deposited on silicon wafers at room temperature under various bias conditions by an off-plane double bend filtered cathodic vacuum arc technique. The microstructural and surface properties of the ta-C:Co films were systematically studied by XPS, Raman, and surface energy measurements via contact angle measurements. Incorporation of Co increases the contact angle sharply to 107.5° as compared to the value for pure ta-C of ∼80°. XPS measurements showed that an increase in negative substrate bias affects both the sp3 content as well as the Co oxidation state on the sample surface. Correlation between XPS and surface energy measurements showed that two different mechanisms might cause the overall decrease in surface free energy of the films.
Characteristics of secondary etching of SiO2 by ions reflected from a primary SiO2 target in a CHF3 plasma
Authors: Lee Gyeo-Re , Hwang Sung-Wook , Min Jae-Ho , Moon Sang Heup
Published: February 2003

The etching of a secondary SiO2 target, target (2), by ions reflected from a primary SiO2 target, target (1), in a CHF3 plasma using various angles for the ions incident on target (1), θi, was examined. The etch rate of target (2) was enhanced by collision with reflected ions and the extent of etch-rate enhancement was significantly affected by the surface roughness of target (1). The extent and range of secondary etching increased when θi was increased from 60° to 80°. Under the conditions used in this study, the maximum scattering angle for initiating secondary etching was nearly constant, at about 60°, irrespective of θi, when the latter was between 60° and 80°. When θi was increased to 85°, the surface of target (1) was covered with a fluorocarbon polymer layer, which drastically decreased the extent of secondary etching. At θi=85°, the maximum scattering angle was lowered to about 40° and, as a result, the range of secondary etching was reduced.