bluuebunny/crossref_metadata_2025_split huggingface dataset:
Removal of the N‐terminal hexapeptide from human β2‐microglobulin facilitates protein aggregation and fibril formation
Authors: Esposito G. , Michelutti R. , Verdone G. , Viglino P. , ÁNdez H. Hern , Robinson C. V. , Amoresano A. , Piaz F. Dal , Monti M. , Pucci P. , Mangione P. , Stoppini M. , Merlini G. , Ferri G. , Bellotti V. 
Published: February 2009

AbstractThe solution structure and stability of N‐terminally truncated β2‐microglobulin (δN6β2‐m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that δN6β2‐m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full‐length proteins at μM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that δN6β2‐m is significantly less protected than its wild‐type counterpart. Analysis of δN6β2‐m by NMR shows that this loss of protection occurs in β strands I, III, and part of II. At mM concentration gel filtration analysis shows that δN6β2‐m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of β2‐microglobulin was found to have a higher tendency to self‐associate than the intact molecule, and unlike wild‐type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that δN6β2‐m could be a key intermediate of a proteolytic pathway of β2‐microglobulin. Overall, the data suggest that removal of the six residues from the N‐terminus of β2‐microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between β‐strands far removed from this constrain is greatly perturbed.
Evidence for phosphorylation‐dependent conformational changes in methylesterase CheB
Authors: Anand Ganesh S. , Goudreau Paul N. , Lewis J. Kathleen , Stock Ann M. 
Published: February 2009

AbstractEnhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N‐terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation‐dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N‐terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N‐ and C‐terminal domains, leading to stimulation of methylesterase activity.
Comparative model building of interleukin‐7 using interleukin‐4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation
Authors: Cosenza Larry , Murphy John R. , Smith Temple , Rosenbach Andrew , White James V. 
Published: February 2009

AbstractUsing a combination of theoretical sequence structure recognition predictions and experimental disulfide bond assignments, a three‐dimensional (3D) model of human interleukin‐7 (hIL‐7) was constructed that predicts atypical surface chemistry in helix D that is important for receptor activation. A 3D model of hIL‐7 was built using the X‐ray crystal structure of interleukin‐4 (IL‐4) as a template (Walter MR et al., 1992, J Mol Biol. 224: 1075‐1085; Walter MR et al., 1992, J Biol Chem 267:20371–20376). Core secondary structures were constructed from sequences of hIL‐7 predicted to form helices. The model was constructed by superimposing IL‐7 helices onto the IL‐4 template and connecting them together in an up‐up down‐down topology. The model was finished by incorporating the disulfide bond assignments (Cys3, Cys142), (Cys35, Cys130), and (Cys48, Cys93), which were determined by MALDI mass spectroscopy and site‐directed mutagenesis (Cosenza L, Sweeney E, Murphy JR, 1997, J Biol Chem 272:32995–33000). Quality analysis of the hIL‐7 model identified poor structural features in the carboxyl terminus that, when further studied using hydrophobic moment analysis, detected an atypical structural property in helix D, which contains Cys130 and Cys142. This analysis demonstrated that helix D had a hydrophobic surface exposed to bulk solvent that accounted for the poor quality of the model, but was suggestive of a region in IL‐7 that may be important for protein interactions. Alanine (Ala) substitution scanning mutagenesis was performed to test if the predicted atypical surface chemistry of helix D in the hIL‐7 model is important for receptor activation. This analysis resulted in the construction, purification, and characterization of four hIL‐7 variants, hIL‐7 (K121A), hIL‐7(L136A), hIL‐7(K140A), and hIL‐7(W143A), that displayed reduced or abrogated ability to stimulate a murine IL‐7 dependent pre‐B cell proliferation. The mutant hIL‐7(W143A), which is biologically inactive and displaces [125I]‐hIL‐7, is the first reported IL‐7R system antagonist.
The energetics of phosphate binding to a protein complex
Authors: Edgcomb Stephen P. , Baker Brian M. , Murphy Kenneth P. 
Published: February 2009

AbstractThe heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease‐inhibitor complex; thus, the elastase‐ovomucoid system provides a unique opportunity to study phosphate‐protein interactions. We have used isothermal titration calorimetry to investigate this binding, thereby providing one of the few complete thermodynamic characterizations of phosphate interacting with proteins. The binding is characterized by a small favorable ΔG°, a large unfavorable ΔH°, and a positive ΔCp, thermodynamics consistent with the release of water being linked to phosphate binding. These measurements provide insight into the binding of phosphotyrosine containing peptides to SH2 domains by suggesting the energetic consequences of binding phosphate free from other interactions.
Role of Lys335 in the metastability and function of inhibitory serpins
Authors: Im Hana , Yu Myeong‐Hee 
Published: February 2009

AbstractThe native form of inhibitory serpins (serine protease inhibitors) is not in the thermodynamically most stable state but in a metastable state, which is critical to inhibitory functions. To understand structural basis and functional roles of the native metastability of inhibitory serpins, we have been characterizing stabilizing mutations of human α1‐antitrypsin, a prototype inhibitory serpin. One of the sites that has been shown to be critical in stability and inhibitory activity of α1‐antitrypsin is Lys335. In the present study, detailed roles of this lysine were analyzed by assessing the effects of 13 different amino acid substitutions. Results suggest that size and architect of the side chains at the 335 site determine the metastability of α1‐antitrypsin. Moreover, factors such as polarity and flexibility of the side chain at this site, in addition to the metastability, seem to be critical for the inhibitory activity. Substitutions of the lysine at equivalent positions in two other inhibitory serpins, human α1‐antichymotrypsin and human antithrombin III, also increased stability and decreased inhibitory activity toward α‐chymotrypsin and thrombin, respectively. These results and characteristics of lysine side chain, such as flexibility, polarity, and the energetic cost upon burial, suggest that this lysine is one of the structural designs in regulating metastability and function of inhibitory serpins in general.
Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA
Authors: Ferguson Andrew D. , Coulton James W. , Diederichs Kay , Welte Wolfram , Braun Volkmar , Fiedler Hans‐Peter 
Published: February 2009

AbstractOne alternative method for drug delivery involves the use of siderophore‐antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron‐chelating siderophores, can be actively transported across the outer membrane of Gram‐negative bacteria. These “Trojan Horse” antibiotics may prove useful as an efficient means to combat multi‐drug–resistant bacterial infections. Here we present the crystallo‐graphic structures of the natural siderophore‐antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad‐host range antibiotics that consist of a hydroxamate‐type iron‐chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate‐type siderophores, the three‐dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron‐chelating siderophores and offers a structural platform for the rational design of hydroxamate‐type siderophore‐antibiotic conjugates.
Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa
Authors: Brocca Stefania , Alberghina Lilia , Lotti Marina , Persson Mattias , Wehtje Ernst , Adlercreutz Patrick 
Published: February 2009

AbstractSequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N‐linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild‐type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
The 1.9 Å crystal structure of Escherichia coli MurG, a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis
Authors: Ha Sha , Walker Deborah , Shi Yigong , Walker Suzanne 
Published: February 2009

AbstractThe 1.9 Å X‐ray structure of a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis is reported. This enzyme, MurG, contains two α/β open sheet domains separated by a deep cleft. Structural analysis suggests that the C‐terminal domain contains the UDP‐GlcNAc binding site while the N‐terminal domain contains the acceptor binding site and likely membrane association site. Combined with sequence data from other MurG homologs, this structure provides insight into the residues that are important in substrate binding and catalysis. We have also noted that a conserved region found in many UDP‐sugar transferases maps to a β/α/β/α supersecondary structural motif in the donor binding region of MurG, an observation that may be helpful in glycosyltransferase structure prediction. The identification of a conserved structural motif involved in donor binding in different UDP‐sugar transferases also suggests that it may be possible to identify—and perhaps alter—the residues that help determine donor specificity.
Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: A quantitative model
Authors: Schulz Tanja , Pleiss Jürgen , Schmid Rolf D. 
Published: February 2009

AbstractThe lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast‐reacting enantiomer in a productive mode and the slow‐reacting enantiomer in a nonproductive mode. Nonproductive mode of fast‐reacting enantiomers was prohibited by repulsive interactions. For the slow‐reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(HNϵ – Oalc) between the histidine side chain and the alcohol oxygen increases. d(HNϵ – Oalc) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(HNϵ – Oalc) is>2.2 Å for slow‐reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(HNϵ – Oalc) is less than 2.0 Å, and slow‐ and fast‐reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E< 100), d(HNϵ – Oalc) is around 2.1 Å. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.
A new model for 20‐hydroxyecdysone and dibenzoylhydrazine binding: A homology modeling and docking approach
Authors: Wurtz Jean‐Marie , Guillot Benoit , Fagart Jérôme , Moras Dino , Tietjen Klaus , Schindler Michael 
Published: February 2009

AbstractThe ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand‐binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20‐hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N‐tert‐butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR–LBD protein models rationalize how 20E and dibenzoylhy‐drazines interact with the ligand‐binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides.
Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein
Authors: GrießLer Richard , D'Auria Sabato , Tanfani Fabio , Nidetzky Bernd 
Published: February 2009

AbstractStarch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5′‐phosphate as an active‐site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500‐fold stabilization against irreversible inactivation at elevated temperatures (≥50°C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate≈︁16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the α‐helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5′‐phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature‐dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to≈︁ 50°C, and (2) stabilizes the covalent protein cofactor linkage up to ≈︁ 70°C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50°C of ≈︁ 25 mM. The extra stability conferred by oxyanion‐ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a ≈︁ 20°C higher value on the temperature scale.
Cascaded multiple classifiers for secondary structure prediction
Authors: Ouali Mohammed , King Ross D. 
Published: February 2009

AbstractWe describe a new classifier for protein secondary structure prediction that is formed by cascading together different types of classifiers using neural networks and linear discrimination. The new classifier achieves an accuracy of 76.7% (assessed by a rigorous full Jack‐knife procedure) on a new nonredundant dataset of 496 nonhomologous sequences (obtained from G.J. Barton and JA. Cuff). This database was especially designed to train and test protein secondary structure prediction methods, and it uses a more stringent definition of homologous sequence than in previous studies. We show that it is possible to design classifiers that can highly discriminate the three classes (H, E, C) with an accuracy of up to 78% for β‐strands, using only a local window and resampling techniques. This indicates that the importance of long‐range interactions for the prediction of β‐strands has been probably previously overestimated.
The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G
Authors: Seewald Michael J. , Pichumani Kumar , Stowell Cheri , Tibbals Benjamin V. , Regan Lynne , Stone Martin J. 
Published: February 2009

AbstractThe contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR‐derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross‐relaxation rates (ηxy), and steady state {1H}‐15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50°C for 89–100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model‐free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ∼9°C lower (78°C rather than 87°C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.
Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles
Authors: Sanghera Narinder , Pinheiro Teresa J.T. 
Published: February 2009

AbstractBinding of native cyt c to L‐PG micelles leads to a partially unfolded conformation of cyt c. This micelle‐bound state has no stable tertiary structure, but remains as α‐helical as native cyt c in solution. In contrast, binding of the acid‐unfolded cyt c to L‐PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L‐PG micelles. Far‐ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle‐associated helical state (HL) has a native‐like α‐helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near‐UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid‐unfolded cyt c was investigated by stopped‐flow tryptophan fluorescence. Formation of HL from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle‐induced unfolding of cyt c occurs at a rate ∼0.1 s∽1, which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid‐unfolded cyt c with L‐PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.