bluuebunny/crossref_metadata_2025_split huggingface dataset:
The binding of myristoylated N‐terminal nonapeptide from neuron‐specific protein CAP‐23/NAP‐22 to calmodulin does not induce the globular structure observed for the calmodulin—nonmyristoylated peptide complex
Authors: Hayashi Nobuhiro , Izumi Yoshinobu , Titani Koiti , Matsushima Norio 
Published: February 2009

AbstractCAP‐23/NAP‐22, a neuron‐specific protein kinase C substrate, is Nα‐myristoylated and interacts with calmodulin (CaM) in the presence of Ca2+ ions. Takasaki et al. (1999, J Biol Chem 274:11848‐11853) have recently found that the myristoylated N‐terminal nonapeptide of CAP‐23/NAP‐22 (mC/N9) binds to Ca2+‐bound CaM (Ca2+/CaM). In the present study, small‐angle X‐ray scattering was used to investigate structural changes of Ca2+/CaM induced by its binding to mC/N9 in solution. The binding of one mC/N9 molecule induced an insignificant structural change in Ca2+/CaM. The 1:1 complex appeared to retain the extended conformation much like that of Ca2+/CaM in isolation. However, it could be seen that the binding of two mC/N9 molecules induced a drastic structural change in Ca2+/CaM, followed by a slight structural change by the binding of more than two but less than four mC/N9 molecules. Under the saturated condition (the molar ratio of 1:4), the radius of gyration (Rg) for the Ca2+/CaM‐mC/N9 complex was 19.8 ± 0.3 Å. This value was significantly smaller than that of Ca2+/CaM (21.9 ± 0.3 Å), which adopted a dumbbell structure and was conversely 2‐3 Å larger than those of the complexes of Ca2+/CaM with the nonmyristoylated target peptides of myosin light chain kinase or CaM kinase II, which adopted a compact globular structure. The pair distance distribution function had no shoulder peak at around 40 Å, which was mainly due to the dumbbell structure. These results suggest that Ca2+/CaM interacts with Nα‐myristoylated CAP‐23/NAP‐22 differently than it does with other nonmyristoylated target proteins. The N‐terminal amino acid sequence alignment of CAP‐23/NAP‐22 and other myristoylated proteins suggests that the protein myristoylation plays important roles not only in the binding of CAP‐23/NAP‐22 to Ca2+/CaM, but also in the protein‐protein interactions related to other myristoylated proteins.
Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI
Authors: Goedken Eric R. , Keck James L. , Berger James M. , Marqusee Susan 
Published: February 2009

AbstractProteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy, we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo‐form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three‐state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.
Lipoylating and biotinylating enzymes contain a homologous catalytic module
Authors: Reche Pedro A. 
Published: February 2009

AbstractBiotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of these moieties to the biotinyl‐ and lipoyl‐dependent enzymes is post‐translationally catalyzed by specific biotinylating and lipoylating protein enzymes. In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation of the relevant enzymes, whereas only one enzyme, the BirA protein, is responsible for all the biotinylation. Counterparts of the E. coli BirA, LplA, and LipB enzymes have been previously identified in many organisms, but homology among the three families has never been reported. Computational analysis based on PSI‐BLAST profiles and secondary structure predictions indicates, however, that lipoylating and biotinylating enzymes are evolutionarily related protein families containing a homologous catalytic module. Sequence conservation among the three families is very poor, but a single lysine residue is strictly conserved in all of them, which, according to the available X‐ray crystal structure of the E. coli BirA protein, is expected to contribute to the binding of lipoic acid in the LplA and LipB enzymes.
Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin
Authors: Galea Charles A. , Dalrymple Brian P. , Kuypers Ron , Blakeley Robert 
Published: February 2009

AbstractThe substrate specificity of porcine pepsin has been altered by site‐directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys‐Pro‐Xaa‐Yaa‐Phe*Nph‐Arg‐Leu (where Xaa is Ile or Pro, Yaa is Glu, Leu, Gln or Lys, Nph is p‐nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two‐ to fourfold higher kcat/Km, compared with wild‐type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild‐type pepsin cleaved a peptide corresponding to the carboxy‐terminal telopeptide region of bovine type I collagen α1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp‐Leu, Leu‐Ser, and Phe‐Leu) and at a significantly lower rate at Ser‐Phe. However, Thr222Val/Glu287Met cleaved site Ser‐Phe at a rate 20‐fold higher than the wild‐type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu‐Ser (similar to cathepsin D) and at a rate 23‐fold higher than the wild‐type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.
Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy
Authors: Bouchard Mario , Zurdo Jesús , Nettleton Ewan J. , Dobson Christopher M. , Robinson Carol V. 
Published: February 2009

AbstractFourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature‐induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native‐like α‐helical structure. On heating to 70°C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT‐IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from α‐helical to β‐sheet structure. At later stages, EM shows the development of fibrils with well‐defined repetitive morphologies including structures with a periodic helical twist of ∼450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.
Comparison of binding energies of SrcSH2‐phosphotyrosyl peptides with structure‐based prediction using surface area based empirical parameterization
Authors: Henriques Denise A. , Ladbury John E. , Jackson Richard M. 
Published: February 2009

AbstractThe prediction of binding energies from the three‐dimensional (3D) structure of a protein–ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent‐accessible surface area‐based calculations for the prediction of the binding of Src‐SH2 domain with a series of tyrosyl phosphopeptides based on the high‐affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+3 position has been changed. Two other peptides based on the C‐terminal regulatory site of the Src protein and the platelet‐derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area‐based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (ΔCp, ΔS, ΔH, and ΔG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent‐accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters; however, the tolerance of this approach is not sufficient to make it ubiquitously applicable.
Protein renaturation by the liquid organic salt ethylammonium nitrate
Authors: Summers Catherine A. , Flowers Robert A. 
Published: February 2009

AbstractThe room‐temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured‐reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one‐step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded‐active protein can be separated from the molten salt by simple desalting methods. Although the use of a low‐temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility.
Purification and refolding of vascular endothelial growth factor‐B
Authors: Scrofani Sergio D.B. , Fabri Louis J. , Xu Ping , Maccarone Pino , Nash Andrew D. 
Published: February 2009

AbstractVascular endothelial growth factor (VEGF)‐A interacts with the receptor tyrosine kinases VEGF‐R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF‐A include VEGF‐B, VEGF‐C, VEGF‐D, and placenta growth factor (PLGF). Compared with VEGF‐A, relatively little is known about the biological role of the VEGF‐R1 specific ligand, VEGF‐B. Two splice variant isoforms that differ at the COOH‐terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development. Recent analysis of mice with a targeted deletion of the VEGF‐B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29‐E35). To facilitate further characterization of VEGF‐B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs). The approach developed resolves a number of significant issues associated with VEGF‐B, including the ability to heterodimerize with endogenous VEGF‐A when co‐expressed in mammalian cells, a complex secondary structure incorporating inter‐ and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins. The resulting purified disulfide‐linked homodimer was demonstrated to bind to VEGF‐R1 and to compete with VEGF‐A for binding to this receptor.
For the record: The three‐dimensional structure of the ternary complex of Corynebacterium glutamicum diaminopimelate dehydrogenase‐NADPH‐L‐2‐amino‐6‐methylene‐pimelate
Authors: Cirilli Maurizio , Scapin Giovanna , Sutherland Andrew , Vederas John C. , Blanchard John S. 
Published: February 2009

AbstractThe three‐dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate d‐dehydrogenase in a ternary complex with NADPH and l‐2‐amino‐6‐methylene‐pimelate has been solved and refined to a resolution of 2.1 Å. l‐2‐Amino‐6‐methylene‐pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 μM) vs. meso‐diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+‐dependent oxidation of the d‐amino acid stereocenter of meso‐diaminopimelate, and is the only enzyme known to catalyze the oxidative deamination of a d‐amino acid. The enzyme is involved in the biosynthesis of meso‐diaminopimelate and l‐lysine from l‐aspartate, a biosynthetic pathway of considerable interest because it is essential for growth of certain bacteria. The dehydrogenase is found in a limited number of species of bacteria, as opposed to the alternative succinylase and acetylase pathways that are widely distributed in bacteria and plants. The structure of the ternary complex reported here provides a structural rationale for the nature and potency of the inhibition exhibited by the unsaturated l‐2‐amino‐6‐methylene‐pimelate against the dehydrogenase. In particular, we compare the present structure with other structures containing either bound substrate, meso‐diaminopimelate, or a conformationally restricted isoxazoline inhibitor. We have identified a significant interaction between the α‐l‐amino group of the unsaturated inhibitor and the indole ring of Trp144 that may account for the tight binding of this inhibitor.
Primary structure of a novel subunit in ba3‐cytochrome oxidase from thermus thermophilus
Authors: Soulimane Tewfik , Buse Gerhard , Dewor Manfred , Than Manuel E. , Huber Robert 
Published: February 2009

AbstractThe ba3‐type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix “subunit IIa” spanning the membrane. The hydrophobic N‐terminally blocked protein was isolated in high yield using high‐performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C‐terminal sequencing of the native protein.The molecular mass of 3,794 Da as determined by MALDI‐MS and by ESI requires the N‐terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5‐Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3‐cytochrome oxidase from Natronobacterium pharaonis.The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).
Integrity of thermus thermophilus cytochrome c552 Synthesized by escherichia coli cells expressing the host‐specific cytochrome c maturation genes, ccmABCDEFGH: Biochemical, spectral, and structural characterization of the recombinant protein
Authors: Fee James A. , Chen Ying , Todaro Thomas R. , Bren Kara L. , Patel Kirti M. , Hill Michael G. , Gomez‐Moran Ester , Loehr Thomas M. , Ai Jingyuan , Thöny‐meyer Linda , Williams Pamela A. , Sturam Enrico , Sridhar Vandana , Mcree Duncan E. 
Published: February 2009

AbstractWe describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid‐borne, chimeric cycA gene encoding an Escherichia coli‐compatible, N‐terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid‐borne, host‐specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme‐copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H‐NMR spectroscopies. The 1.7 Å resolution X‐ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629–644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.
Structure of the Ca2+‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure
Authors: Liu Zhi‐Jie , Vysotski Eugene S. , Vysotski Eugene S. , Chen Chun‐Jung , Rose John P. , Lee John , Wang Bi‐Cheng 
Published: February 2009

AbstractThe crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 Å resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2‐position. The protein‐coelenterazine 2‐oxy complex observed in the crystals is photo‐active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the second de novo protein structure determined using the anomalous scattering signal of the sulfur substructure in the crystal. The method used here is theoretically different from that used for crambin in 1981 (4.72 kDa) and represents a significant advancement in protein crystal structure determination.
Fragment complementation of calbindin D28k
Authors: Berggård Tord , Thulin Eva , Linse Sara , Åkerfeldt Karin S. 
Published: February 2009

AbstractCalbindin D28k is a highly conserved Ca2+‐binding protein abundant in brain and sensory neurons. The 261‐residue protein contains six EF‐hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF‐hands each (residues 1–132 and 133–261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion‐exchange and size‐exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF‐hands 1, 2, 3, or 4. The C‐terminal half (residues 133–261) appears to have a lower intrinsic stability compared to the N‐terminal half (residues 1–132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1–132 and 133–261 fragments. The affinity between these two fragments is 2 × 107 M−1, with association and dissociation rate constants of 2.7 × 104 M−1 s−1 and 1.4 × 10−3 s−1, respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual α‐helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The α‐helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+‐induced folding of the fragments is important for successful reconstitution of calbindin D28k.
Internal motional amplitudes and correlated bond rotations in an α‐helical peptide derived from 13C and 15N NMR relaxation
Authors: Idiyatullin Djaudat , Krushelnitsky Alexei , Nesmelova Irina , Daragan Vladimir A. , Mayo Kevin H. , Blanco Francesco , Serrano Luis 
Published: February 2009

AbstractPeptide GFSKAELAKARAAKRGGY folds in an α‐helical conformation that is stabilized by formation of a hydrophobic staple motif and an N‐terminal capping box (Munoz V, Blanco FJ, Serrano L, 1995, Struct Biol 2:380–385). To investigate backbone and side‐chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C‐ and 15N‐enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20° and backbone ϕ,ψ bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Cα‐H, Cα‐Cβ, and N‐H rocking‐type motions along the helix dipole axis. L7 side‐chain CβH2 and CγH motions are also correlated and as motionally restricted as backbone CαH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling‐in‐a‐cone type motions dominate in water, whereas in TFE, the Cβ‐Cγ bond and phenyl ring fluctuate more simply about the Cα‐Cβ bond. These data support the Daragan‐Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378–8388) and contribute to a general understanding of internal motions in peptides and proteins.