bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of wheat α‐thionin with large unilamellar vesicles
Authors: Caaveiro José Manuel M. , Goñi Félix M. , González‐Mañas Juan Manuel , Molina Antonio , Rodriguez‐Palenzuela Pablo 
Published: February 2009

AbstractThe interaction of the wheat antibacterial peptide α‐thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat α‐fhionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine‐poly(ethylene glycol 2000) (PEG‐PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8‐aminonaphthalene‐1,3,6, trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.
Empirical solvent‐mediated potentials hold for both intra‐molecular and inter‐molecular inter‐residue interactions
Authors: Keskin O. , Bahar I. , Jernigan R. L. , Badretdinov A. Y. , Ptitsyn O. B. 
Published: February 2009

AbstractWhether knowledge‐based intra‐molecular inter‐residue potentials are valid to represent inter‐molecular interactions taking place at protein‐protein interfaces has been questioned in several studies. Differences in the chain connectivity effect and in residue packing geometry between interfaces and single chain monomers have been pointed out as possible sources of distinct energetics for the two cases. In the present study, the interfacial regions of protein‐protein complexes are examined to extract inter‐molecular inter‐residue potentials, using the same statistical methods as those previously adopted for intra‐molecular residue pairs. Two sets of energy parameters are derived, corresponding to solvent‐mediation and “average residue” mediation. The former set is shown to be highly correlated (correlation coefficient 0.89) with that previously obtained for inter‐residue interactions within single chain monomers, while the latter exhibits a weaker correlation (0.69) with its intra‐molecular counterpart. In addition to the close similarity of intra‐ and inter‐molecular solvent‐mediated potentials, they are shown to be significantly more residue‐specific and thereby discriminative compared to the residue‐mediated ones, indicating that solvent‐mediation plays a major role in controlling the effective inter‐residue interactions, either at interfaces, or within single monomers. Based on this observation, a reduced set of energy parameters comprising 20 one‐body and 3 two‐body terms is proposed (as opposed to the 20 × 20 tables of inter‐residue potentials), which reproduces the conventional 20 × 20 tables with a correlation coefficient of 0.99.
Domain structure analysis of elongation factor‐3 from saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry
Authors: Ladror Uri S. , Egan David A. , Snyder Seth W. , Capobianco John O. , Goldman Robert C. , Dorwin Sarah A. , Johnson Robert W. , Edalji Rohinton , Sarthy Aparna V. , Mcgonigal Tom , Walter Karl A. , Holzman Thomas F. 
Published: February 2009

AbstractElongation‐factor‐3 (EF‐3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF‐3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultra‐centrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 °C, and a minor transition at 58 °C. Ultracentrifugation shows that EF‐3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF‐3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF‐3 with trypsin produced the first cleavage at the N‐side of Gln775, generating a 90‐kDa N‐terminal fragment and a 33‐kDa C‐terminal fragment. The N‐terminal fragment slowly undergoes further digestion generating two major bands, one at ∼75 kDa and the other at ∼55 kDa. The latter was unusually resistant to further tryptic digestion. The 33‐kDa C‐terminal fragment was highly sensitive to tryptic digestion. A 30‐min tryptic digest showed that the N‐terminal 60% of EF‐3 was relatively inaccessible to trypsin, whereas the C‐terminal 40% was readily digested. These results suggest a tight structure of the N‐terminus, which may give rise to the 58 °C transition, and a loose structure of the C‐terminus, giving rise to the 39 °C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5‐homologous domain (Lys102‐Ile368), the N‐terminal ATP‐binding cassette (Gly463‐Lys622), and the aminoacyl‐tRNA‐synthase homologous domain (Glu820‐Gly865). Both the basal and ribosome‐stimulated ATPase activities were inactivated by trypsin, but the ribosome‐stimulated activity was inactivated faster.
Localization of basic residues required for receptor binding to the single α‐helix of the receptor binding domain of human α2‐macroglobulin
Authors: Huang Wen , Dolmer Klavs , Liao Xiubei , Gettins Peter G. W. 
Published: February 2009

AbstractTo better understand the structural basis for the binding of proteinase‐transformed human α2‐macroglobulin (α2M) to its receptor, we have used three‐dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human α2M. Assignment of the backbone NMR resonances of RBD was made using 13C/15N and 15N‐enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter‐ and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in β‐conformation and one α‐helix, which together comprise 44% of the protein. The β‐strands form three regions of antiparallel β‐sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the α‐helix, which also contains an (Arg1378). Secondary structure predictions of other α‐macroglobulins show the conservation of this α‐helix and suggest an important role for this helix and for basic residues within it for receptor binding.
Self‐organizing tree‐growing network for the classification of protein sequences
Authors: Fraga Luis Gerardo De La , Carazo Jose Maria , Wang Huai‐Chun , Dopazo Joaquin , Zhu Yun‐Ping 
Published: February 2009

AbstractThe self‐organizing tree algorithm (SOTA) was recently introduced to construct phylogenetic trees from biological sequences, based on the principles of Kohonen's self‐organizing maps and on Fritzke's growing cell structures. SOTA is designed in such a way that the generation of new nodes can be stopped when the sequences assigned to a node are already above a certain similarity threshold. In this way a phylogenetic tree resolved at a high taxonomic level can be obtained. This capability is especially useful to classify sets of diversified sequences. SOTA was originally designed to analyze pre‐aligned sequences. It is now adapted to be able to analyze patterns associated to the frequency of residues along a sequence, such as protein dipeptide composition and other n‐gram compositions. In this work we show that the algorithm applied to these data is able to not only successfully construct phylogenetic trees of protein families, such as cytochrome c, triosephophate isomerase, and hemoglobin alpha chains, but also classify very diversified sequence data sets, such as a mixture of interleukins and their receptors.
Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct
Authors: Chen Guo‐Qiang , Sun Yu , Jin Rongsheng , Gouaux Eric 
Published: February 2009

AbstractIonotropic glutamate receptors constitute an important family of ligand‐gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA‐sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N‐terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997.Proc Natl Acad Sci USA 94:13431–13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S2I construct proved to be trypsin‐ and chymotrypsin‐resistant, stable to storage at 4 °C, and amenable to three‐dimensional crystal formation. The HS1S2I variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 Å resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.
Effect of denaturant and protein concentrations upon protein refolding and aggregation: A simple lattice model
Authors: Gupta Prashant , Hall Carol K. , Voegler Anne C. 
Published: February 2009

AbstractWe present a study of the competition between protein refolding and aggregation for simple lattice model proteins. The effect of solvent conditions (i.e., the denaturant concentration and the protein concentration) on the folding and aggregation behavior of a system of simple, two‐dimensional lattice protein molecules has been investigated via dynamic Monte Carlo simulations. The population profiles and aggregation propensities of the nine most populated intermediate configurations exhibit a complex dependence on the solution conditions that can be understood by considering the competition between intra‐ and interchain interactions. Some of these configurations are not even seen in isolated chain simulations; they are observed to be highly aggregation prone and are stabilized primarily by the aggregation reaction in multiple‐chain systems. Aggregation arises from the association of partially folded intermediates rather than from the association of denatured random‐coil states. The aggregation reaction dominates over the folding reaction at high protein concentration and low denaturant concentration, resulting in low refolding yields at those conditions. However, optimum folding conditions exist at which the refolding yield is a maximum, in agreement with some experimental observations.
Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase
Authors: Lorenzo Claudia De , Piccoli Renata , Maro Antimo Di , Pucci Piero , D'alessio Giuseppe , Piaz Fabrizio Dal 
Published: February 2009

AbstractDimeric seminal RNase (BS‐RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N‐terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N‐terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N‐terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface.
Solution structure of the His 12 → Cys mutant of the N‐terminal zinc binding domain of HIV‐1 integrase complexed to cadmium
Authors: Cai Mengli , Caffrey Michael , Clore G. Marius , Gronenborn Angela M. , Huang Ying , Zheng Ronglan , Craigie Robert 
Published: February 2009

AbstractThe solution structure of His 12 → Cys mutant of the N‐terminal zinc binding domain (residues 1–55; IN1–55) of HIV‐1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild‐type N‐terminal domain complexed to zinc. In contrast to the wild‐type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His 12 → Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10–18 is intermediate between the two forms of the wild‐type complex.
Ca2+‐dependent conformational changes in bovine GCAP‐2
Authors: Hughes Robert E. , Hurley James B. , Brzovic Peter S. , Dizhoor Alexander M. , Klevit Rachel E. 
Published: February 2009

AbstractGCAP‐2, a mammalian photoreceptor‐specific protein, is a Ca2+‐dependent regulator of the retinal membrane guanylyl cyclases (Ret‐GCs). Sensing the fall in intracellular free Ca2+ after photo‐excitation, GCAP‐2 stimulates the activity of Ret‐GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP‐2 is a small N‐myristoylated protein containing four EF‐hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP‐2 alters its conformation in response to Ca2+‐binding as measured by a Ca2+‐dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+‐bound and Ca2+‐free forms of GCAP‐2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N‐terminus of Ca2+‐bound GCAP‐2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N‐terminus of GCAP‐2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP‐2 and recoverin, the structural consequences of Ca2+‐binding for these two proteins are significantly dissimilar.
A solution SAXS study of borrelia burgdorferi OspA, a protein containing a single‐layer β‐sheet
Authors: Bu Zimei , Engelman Donald M. , Koide Shohei 
Published: February 2009

AbstractThe crystal structure of a soluble form of Borrelia burgdorferi outer surface protein A (OspA) complexed with the Fab fragment of a monoclonal antibody has revealed an unusual structure that has a repetitive antiparallel β topology with a nonglobular, single layer β‐sheet connecting the globular N‐ and C‐terminal domains. Earlier NMR studies have shown that the local structure of OspA including the single layer β‐sheet is similar to the crystal structure. Here we report a small angle X‐ray scattering (SAXS) study of the global conformation of OspA in solution. The radius of gyration (Rg) and the length distribution function (P(r)) of OspA measured by SAXS in solution are nearly identical to the calculated ones from the crystal structure, respectively. The NMR and SAXS experiments complement each other to show that OspA including the central single‐layer β‐sheet is a stable structure in solution, and that the OspA crystal structure represents the predominant solution conformation of the protein.
Yeast methionine aminopeptidase I can utilize either Zn2+ or Co2+ as a cofactor: A case of mistaken identity?
Authors: Walker Kenneth W. , Bradshaw Ralph A. 
Published: February 2009

AbstractYeast methionine aminopeptidase I (MetAP I) is one of two enzymes in Saccharomyces cerevisiae that is responsible for cotranslational cleavage of initiator methionines. It has previously been classified as a Co2+ metalloprotease in all prokaryotic and eukaryotic forms studied. However, treatment of recombinant apo‐MetAP I with 12.5 μM Zn2+ produces an enzyme that is as active as that reconstituted with 200 μM Co2+. In the presence of physiological concentrations of reduced glutathione (GSH), Co‐MetAP I is inactive, while the activity of Zn‐MetAP I is increased more than 1.7‐fold over Zn‐MetAP I assayed in the absence of GSH. Given that the in vivo concentration of Zn2+ is at least 1,000‐fold higher than that of Co2+, and that Co2+ is insoluble in physiological concentrations of GSH, it is probable that yeast MetAP I is actually a Zn2+ metalloprotease. Furthermore, unless there are extraordinary conditions that insulate or sequester them from this reducing milieu, that have yet to be identified, there are not likely to be any cytoplasmic enzymes that use free Co2+.
Re‐examining the role of Lys67 in class C β‐lactamase catalysis
Authors: Chen Yu , McReynolds Andrea , Shoichet Brian K. 
Published: February 2009

AbstractLys67 is essential for the hydrolysis reaction mediated by class C β‐lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C β‐lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2‐ to 3‐fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C β‐lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61‐fold decrease in kcat, the deacylation rate. We have determined the structure of the K67R mutant by X‐ray crystallography both in apo and transition state‐analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C β‐lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.
A review about nothing: Are apolar cavities in proteins really empty?
Authors: Matthews Brian W. , Liu Lijun 
Published: February 2009

AbstractCavities within proteins that are strictly apolar typically appear to be empty. It has been suggested, however, that water molecules may be present within such cavities but are too disordered to be seen in conventional crystallographic analyses. In contrast, it is argued here that solvent mobility will be limited by the size of the cavity and for this reason high‐occupancy solvent in cavities of typical volume should be readily detectable using X‐ray crystallography. Recent experimental studies of cavity hydration are reviewed. Such studies are consistent with theoretical predictions that it is energetically unfavorable to have a single water molecule in an apolar cavity. As apolar cavities become larger, a point is reached where it is favorable to have the cavity occupied by a cluster of mutually H‐bonded water molecules. The exact size of such a cavity in a protein is yet to be verified.
Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases
Authors: Zani Marie‐Louise , Baranger Kévin , Guyot Nicolas , Dallet‐Choisy Sandrine , Moreau Thierry 
Published: February 2009

AbstractThe secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar Kis, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.
The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C‐terminal juxtamembrane residues
Authors: Peng Weng Chuan , Lin Xin , Torres Jaume 
Published: February 2009

AbstractThe fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of α and β integrin TM interactions. However, we show herein that in FGFR3‐TM, four C‐terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3‐TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C‐terminal residues were present. In the absence of these four residues, A391E was dimer‐destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3‐TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR‐TM.