bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional consequences of mutations at the allosteric interface in hetero‐ and homo‐hemoglobin tetramers
Authors: Baudin V. , Pagnier J. , Kiger L. , Kister J. , Bihoreau M. T. , Lacaze N. , Marden M. C. , Poyart C. , Schaad O. , Edelstein S. J. 
Published: February 2009

AbstractA seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While α chains associate weakly into dimers, β chains self‐associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the β4 tetramer is frozen in a quaternary R‐state resembling that of liganded HbA. X‐ray diffraction studies of the liganded β4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the “allosteric” interface (α1β2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T‐state of the β4 tetramer. We have studied natural and artificial Hb mutants at different sites in the β chains responsible for the T‐state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme‐heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero‐ and homotetramers. Our conclusion is that the “allosteric” interface is so precisely tailored for maintaining the assembly between αβ dimers that any change in the side chains of β40 (C6), β99 (G1), and β101 (G3) involved in the interface results in increased R‐state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the β4 hemoglobin and the formation of lower affinity noncooperative monomers.
Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis
Authors: Hess Daniel , Winz Robert , Brownsey Roger W. , Aebersold Ruedi , Covey Tom C. 
Published: February 2009

AbstractWe report the use of microbore reverse‐phase high performance liquid chromatography connected on‐line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow‐splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer.The method was used to analyze the 265‐kDa and the 280‐kDa isoforms of the enzyme acetyl‐CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error‐free data interpretation in chemical high‐sensitivity peptide sequence analysis.
Hematopoietic cytokines: Similarities and differences in the structures, with implications for receptor binding
Authors: Wlodawer Alexander , Pavlovsky Alexander , Gustchina Alla 
Published: February 2009

AbstractCrystal and NMR structures of helical cytokines–interleukin‐4 (IL‐4), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and interleukin‐2 (IL‐2)–have been compared. Root mean square deviations in the Cα coordinates for the conserved regions of the helices were 1–2 Å between different cytokines, about twice the differences observed for independently determined crystal and solution structures of IL‐4. Considerable similarity in amino acid sequence in the areas expected to interact with the receptors was detected, and the available mutagenesis data for these cytokines were correlated with structure conservation. Models of cytokine‐receptor interactions were postulated for IL‐4 based on its structure as well as on the published structure of human growth hormone interacting with its receptors (de Vos, A.M., Ultsch, M., & Kossiakoff, A.A., 1992, Science 255, 306–312). Patches of positively charged residues on the surfaces of helices C and D of IL‐4 may be responsible for the interactions with the negatively charged residues found in the complementary parts of the IL‐4 receptors.
Conformational instability of the N‐ and C‐terminal lobes of porcine pepsin in neutral and alkaline solutions
Authors: Lin Xinli , Loy Jeffrey A. , Sussman Fredy , Tang Jordan 
Published: February 2009

AbstractPepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two‐chain pepsin or a two‐chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257–17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline‐inactivated pepsin was regenerated by the addition of the recombinant N‐terminal lobe but not by the C‐terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N‐terminal lobe. A complex between recombinant N‐terminal lobe of pepsinogen and alkaline‐denatured pepsin has been isolated. This complex is structurally similar to a two‐chain pepsinogen, but it contains an extension of a denatured pepsin N‐terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N‐terminal lobe. The structural components that are responsible for the alkaline instability of the N‐terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N‐terminal domain (as compared to 19 in the C‐terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118. Especially, the partially buried Asp11, which interacts with Asp159, could cause one of these two groups to have an abnormally high pKa and the other an abnormally low pKa value. Thus, the ionization of Asp11 at a high pH may place two negatively charged residues in close vicinity. This unfavorable situation may be the trigger for the denaturation of the N‐terminal lobe of pepsin.
The structure and function of omega loop A replacements in cytochrome c
Authors: Murphy Michael E. P. , Brayer Gary D. , Fetrow Jacquelyn S. , Burton Randall E. 
Published: February 2009

AbstractThe structural and functional consequences of replacing Ω‐loop A (residues 18–32) in yeast iso‐1‐cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three‐dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X‐ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild‐type yeast iso‐1‐cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of Ω‐loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, Ω‐loop A contains a valine at position 20, which restores the original wild‐type packing arrangement of the hydrophobic core. Also, as a result of Ω‐loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main‐chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild‐type yeast iso‐1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 °C. This is 19 mV less than the wild‐type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of Ω‐loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.
Interactions and inhibition of blood coagulation factor Va involving residues 311–325 of activated protein C
Authors: Mesters Rolf M. , Heeb Mary J. , Griffin John H. 
Published: February 2009

AbstractActivated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide‐(311–325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa‐1‐stage coagulation assays in normal and in protein S‐depleted plasma with 50% inhibition at 13 μM peptide. In a system using purified clotting factors, peptide‐(311–325) inhibited APC‐catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 μM peptide. However, peptide‐(311–325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]α1‐antitrypsin. Peptide‐(311–325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 μM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose‐dependent manner to immobilized peptide‐(311–325). Peptide‐(311–315) inhibited the binding of factor Va to immobilized APC or factor Xa. These data are consistent with the hypothesis that residues 311–325 in APC bind to factor Va at a site that can bind either APC or factor Xa, and that peptide‐(311–325) interferes with both APC inactivation of factor Va and expression of factor Xa activity in the prothrombinase complex by binding to this site.
Prolyl isomerases catalyze antibody folding in vitro
Authors: Lilie Hauke , Buchner Johannes , Lang Kurt , Rudolph Rainer 
Published: February 2009

AbstractSome slow‐folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl‐peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules. An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased. Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate‐limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalysts of protein folding by accelerating the rate‐limiting isomerization of Xaa‐Pro peptide bonds. Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction. (2) PPI and BSA are able to increase the yield of refolding of Fab by reducing the formation of aggregates or the adsorption to the surface of the reaction vessel in an unspecific manner. This behavior is clearly distinct from the mechanism of action observed with the chaperone GroEL.
Thermodynamics of apocytochrome b5 unfolding
Authors: Pfeil Wolfgang 
Published: February 2009

AbstractApocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two‐state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (ΔG) amounts to ΔG = 7 ± 1 kJ/mol at 25 °C (pH 7.4) compared with ΔG = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, nativelike protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1–35 and 79–90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199–207).
Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations
Authors: Zheng Jianhua , Knighton Daniel R. , Taylor Susan S. , Xuong Nguyen‐Huu , Sowadski Janusz M. , Eyck Lynn F. Ten 
Published: February 2009

AbstractThree crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP‐dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154–2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 Å resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C‐terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active‐site region surrounding the glycine‐rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N‐terminal 40 residues that lie outside the conserved catalytic core are anchored by the N‐terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.
Identification, classification, and analysis of beta‐bulges in proteins
Authors: Chan A. W. Edith , Hutchinson E. Gail , Harris Daniel , Thornton Janet M. 
Published: February 2009

AbstractA β‐bulge is a region of irregularity in a β‐sheet involving two β‐strands. It usually involves two or more residues in the bulged strand opposite to a single residue on the adjacent strand. These irregularities in β‐sheets were identified and classified automatically, extending the definition of β‐bulges given by Richardson et al. (Richardson, J.S., Getzoff, E.D., & Richardson, D.C., 1978, Proc. Natl. Acad. Sci. USA 75, 2574–2578). A set of 182 protein chains (170 proteins) was used, and a total of 362 bulges were extracted. Five types of β‐bulges were found: classic, G1, wide, bent, and special. Their characteristic amino acid preferences were found for most classes of bulges. Basically, bulges occur frequently in proteins; on average there are more than two bulges per protein. In general, β‐bulges produce two main changes in the structure of a β‐sheet: (1) disrupt the normal alternation of side‐chain direction; (2) accentuate the twist of the sheet, altering the direction of the surrounding strands.
Solution structure of the calcium channel antagonist ω‐conotoxin GVIA
Authors: Skalicky Jack J. , Ciesla D. Joseph , Pardi Arthur , Metzler William J. , Galdes Alphonse 
Published: February 2009

AbstractThe three‐dimensional solution structure is reported for ω‐conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen‐bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple‐stranded β‐sheet containing a β‐hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.
Effect of a single aspartate on helix stability at different positions in a neutral alanine‐based peptide
Authors: Huyghues‐Despointes Beatrice M. P. , Scholtz J. Martin , Baldwin Robert L. 
Published: February 2009

AbstractA single aspartate residue has been placed at various positions in individual peptides for which the alanine‐based reference peptide is electrically neutral, and the helix contents of the peptides have been measured by circular dichroism. The dependence of peptide helix content on aspartate position has been used to determine the helix propensity (s‐value). Both the charged (Asp−) and uncharged (Asp0) forms of the aspartate residue are strong helix breakers and have identical s‐values of 0.29 at 0°C. The interaction of Asp− with the helix dipole affects helix stability at positions throughout the helix, not only near the N‐terminus, where the interaction is helix stabilizing, and the C‐terminus, where it is destabilizing. Comparison of the helix contents at acidic pH (Asp0) and at neutral pH (Asp−) shows that the charge–helix dipole interaction is screened slowly with increasing NaCl concentration, and screening is not complete even at 4.8 M NaCl. Lastly, a helix‐stabilizing hydrogen‐bond interaction between glutamine and aspartate (spacing i, i + 4) has been found. This side‐chain interaction is specific for both the orientation and spacing of the glutamine and aspartate residues and is resistant to screening by NaCl.
Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms
Authors: Priestle John P. , Rahuel Joseph , Rink Hans , Tones Michael , Grütter Markus G. 
Published: February 2009

AbstractThe three‐dimensional structures of d‐Phe‐Pro‐Arg‐chloromethyl ketone‐inhibited thrombin in complex with Tyr‐63‐sulfated hirudin55–65 (ternary complex) and of thrombin in complex with the bifunctional inhibitor d‐Phe‐Pro‐Arg‐Pro‐(Gly)4‐hirudin54–65 (CGP 50, 856, binary complex) have been determined by X‐ray crystallography in crystal forms different from those described by Skrzypczak‐Jankun et al. (Skrzypczak‐Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379–1393). In both complexes, the interactions of the C‐terminal hirudin segments of the inhibitors binding to the fibrinogen‐binding exosite of thrombin are clearly established, including residues 60–64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr‐63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10‐fold lower Ki than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146–150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C‐terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor–thrombin complex, the peptide bond between Arg‐Pro (P1‐P1′) seems to be cleaved.
Prediction of protein conformation on the basis of a search for compact structures: Test on avian pancreatic polypeptide
Authors: Liwo A. , Wawak R. J. , Scheraga H. A. , Pincus M. R. , Rackovsky S. 
Published: February 2009

AbstractBased on the concept that hydrophobic interactions cause a polypeptide chain to adopt a compact structure, a method is proposed to predict the structure of a protein. The procedure is carried out in four stages: (1) use of a virtual‐bond united‐residue approximation with the side chains represented by spheres to search conformational space extensively using specially designed interactions to lead to a collapsed structure, (2) conversion of the lowest‐energy virtual‐bond united‐residue chain to one with a real polypeptide backbone, with optimization of the hydrogen‐bond network among the backbone groups, (3) perturbation of the latter structure by the electrostatically driven Monte Carlo (EDMC) procedure, and (4) conversion of the spherical representation of the side chains to real groups and perturbation of the whole molecule by the EDMC procedure using the empirical conformational energy program for peptides (ECEPP/2) energy function plus hydration. Application of this procedure to the 36‐residue avian pancreatic polypeptide led to a structure that resembled the one determined by X‐ray crystallography; it had an α‐helix starting at residue 13, with the N‐terminal portion of the chain in an extended conformation packed against the α‐helix. Similar structures with slightly higher energies, but looser packing, were also obtained.