bluuebunny/crossref_metadata_2025_split huggingface dataset:
Individual amino acids in the N‐terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases
Authors: Welfle Karin , Politz Oliver , Borriss Rainer , Misselwitz Rolf , Welfle Heinz 
Published: February 2009

AbstractThermostability and unfolding behavior of the wild‐type (1,3–1,4)‐β‐glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12‐M)ΔF14 and H(A12‐M)ΔY13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12‐M)ΔF14 is constructed by the fusion of 12 N‐terminal amino acids of AMY with amino acids 13–214 of MAC, and by deletion of F14. In H(A12‐M)ΔY13F14A, the N‐terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala. The sequence of the N‐terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes. The half transition temperatures Tm are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca2+‐free buffer, MAC, H(A12‐M)ΔF14 and H(A12‐M)ΔY13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two‐step unfolding was found. In the presence of Ca2+, the two‐step unfolding of AMY is strengthened. Furthermore, for H(A12‐M)ΔF14, a two‐step unfolding is induced by Ca2+. These data indicate a two‐domain structure of AMY and H(A12‐M)ΔF14 in the presence of Ca2+. Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+.
Characterization of the S1 binding site of the glutamic acid‐specific protease from Streptomyces griseus
Authors: Stennicke Henning R. , Breddam Klaus , Birktoft Jens J. 
Published: February 2009

AbstractThe glutamic acid‐specific protease from Streptomyces griseus (SGPE) is an 18.4‐kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin‐1β converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the — 1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis but has an influence on stabilization of the ground state. From the three‐dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1.
Identification and application of the concepts important for accurate and reliable protein secondary structure prediction
Authors: King Ross D. , Sternberg Michael J.E. 
Published: February 2009

AbstractA protein secondary structure prediction method from multiply aligned homologous sequences is presented with an overall per residue three‐state accuracy of 70.1%. There are two aims: to obtain high accuracy by identification of a set of concepts important for prediction followed by use of linear statistics; and to provide insight into the folding process. The important concepts in secondary structure prediction are identified as; residue conformational propensities, sequence edge effects, moments of hydrophobicity, position of insertions and deletions in aligned homologous sequence, moments of conservation, auto‐correlation, residue ratios, secondary structure feedback effects, and filtering. Explicit use of edge effects, moments of conservation, and auto‐correlation are new to this paper. The relative importance of the concepts used in prediction was analyzed by stepwise addition of information and examination of weights in the discrimination function. The simple and explicit structure of the prediction allows the method to be reimplemented easily. The accuracy of a prediction is predictable a priori. This permits evaluation of the utility of the prediction: 10% of the chains predicted were identified correctly as having a mean accuracy of >80%. Existing high‐accuracy prediction methods are “black‐box” predictors based on complex nonlinear statistics (e.g., neural networks in P.HD: Rost & Sander, 1993a). For medium‐ to short‐length chains (≥90 residues and <170 residues), the prediction method is significantly more accurate (P < 0.01) than the PHD algorithm (probably the most commonly used algorithm). In combination with the PHD, an algorithm is formed that is significantly more accurate than either method, with an estimated overall three‐state accuracy of 72.4%, the highest accuracy reported for any prediction method.
Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange
Authors: Plesniak Leigh A. , Connelly Gregory P. , Mcintosh Lawrence P. , Wakarchuk Warren W. 
Published: February 2009

AbstractBacillus circulansxylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic‐aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the1H,13C, and15N resonances and tautomeric forms of the imidazole rings were obtained from two‐dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded1Hϕ2of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 × 10−5s−1at pH*7.04 and 30°C) is retarded by > 106‐fold relative to an exposed histidine. The pKaof His 156 is unperturbed at ˜6.5, as measured from the pH dependence of the15N‐ and1H‐NMR spectra of BCX. In contrast, His 149 has a pKa< 2.3, existing in the neutral Nϕ2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30°C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.
A novel organic solvent‐based coupling method for the preparation of covalently immobilized proteins on gold
Authors: Parker Marie‐Claire , Patel Nikin , Davies Martyn C. , Roberts Clive J. , Tendler Saul J.B. , Williams Philip M. 
Published: February 2009

AbstractA novel organic solvent‐based coupling method has been developed for the covalent immobilization of biological material to gold surfaces. The method employs the polar organic solvent anhydrous 2,2,2‐trifluoroethanol as the reaction medium and involves dissolution of the protein (catalase) in the solvent allowing protein coupling to proceed under basic conditions in a dry organic environment. The advantage of this method is that protein attachment is favored over hydrolysis of the coupling reagent. We have shown qualitatively and quantitatively that following attachment to the gold surface a significant proportion of the enzyme catalase remains catalytically active (at least 20–31%).
Comparison of the NMR and X‐ray structures of the HIV‐1 matrix protein: Evidence for conformational changes during viral assembly
Authors: Massiah Michael A. , Worthylake David , Christensen Allyson M. , Sundquist Wesley I. , Hill Christopher P. , Summers Michael F. 
Published: February 2009

AbstractThe three‐dimensional solution‐ and solid‐state structures of the human immunodeficiency virus type‐1 (HIV‐1) matrix protein have been determined recently in our laboratories by NMR and X‐ray crystallographic methods (Massiah et al. 1994. J Mol Biol 244:198–223; Hill et al. 1996. Proc Natl Acad Sci USA 93:3099–3104). The matrix protein exists as a monomer in solution at low millimolar protein concentrations, but forms trimers in three different crystal lattices. Although the NMR and X‐ray structures are similar, detailed comparisons have revealed an approximately 6 Å displacement of a short 310 helix (Pro 66∼Gly 71) located at the trimer interface. High quality electron density and nuclear Overhauser effect (NOE) data support the integrity of the X‐ray and NMR models, respectively. Because matrix apparently associates with the viral membrane as a trimer, displacement of the 310 helix may reflect a physiologically relevant conformational change that occurs during virion assembly and disassembly. These findings further suggest that Pro 66 and Gly 71, which bracket the 310 helix, serve as “hinges” that allow the 310 helix to undergo this structural reorientation.
Activator anion binding site in pyridoxal phosphorylase b: The binding of phosphite, phosphate, and fluorophosphate in the crystal
Authors: Oikonomakos Nikos G. , Zographos Spyros E. , Tsitsanou Katerina E. , Johnson Louise N. , Acharya K. Ravi 
Published: February 2009

AbstractIt has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5′‐phosphate (Chang YC, McCalmont T, Graves DJ. 1983. Biochemistry 22:4987–4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X‐ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R‐state‐like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T‐state inhibitor glucose. Co‐crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P43212, with native‐like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5–19.2%, for data between 8 and 2.4 Å resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5′‐phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 Å). The structural results show that the structures of the pyridoxal phosphorylase b‐anion‐glucose‐IMP complexes are overall similar to the glucose complex of native T‐state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.
Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus
Authors: Prasad G. Sridhar , Stura E.A. , Mcree D.E. , Laco G.S. , Hasselkus‐Light C. , Elder J.H. , Stout C.D. 
Published: February 2009

AbstractWe have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 Å resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P63crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity toE. colidUTPase. In both enzymes the C‐terminal strand of an anti‐parallel β‐barrel participates in the β‐sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P63crystal form one trimer packs on the 63 screw‐axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C‐terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol‐encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.
Significant hydrogen exchange protection in GroEL‐bound DHFR is maintained during iterative rounds of substrate cycling
Authors: Groß Michael , Robinson Carol V. , Mayhew Mark , Hartl F. Ulrich , Radford Sheena E. 
Published: February 2009

AbstractAn unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP‐driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATR Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL‐assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.
The ubiquitous cofactor NADH protects against substrate‐induced inhibition of a pyridoxal enzyme
Authors: Jones Wanda M. , Van Ophem Peter W. , Pospischil Maria A. , Ringe Dagmar , Petsko Gregory , Soda Kenji , Manning James M. 
Published: February 2009

AbstractIn the usual reaction catalyzed by D‐amino acid transaminase, cleavage of the alpha‐H bond is followed by the reversible transfer of the alpha‐NH2 to a keto acid cosubstrate in a two‐step reaction mediated by the two vitamin B6 forms pyridoxal 5′‐phosphate (PLP) and pyridoxamine 5′‐phosphate (PMP). We report here a reaction not on the main pathway, i.e., β‐decarboxylation of D‐aspartate to D‐alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, β‐C‐C bond cleavage of the single substrate D‐aspartate occurs rather than the usual α‐bond cleavage in the transaminase reaction. The D‐alanine produced from D‐aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D‐aspartate turnover and D‐alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzymeD‐aspartate complex with an apparent dissociation constant of 16 μM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of β‐decarboxylase activity by NADH occurs concomitant with a reduction in the 415‐nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. α‐Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 μM. In addition to its known role in shuttling electrons in oxidation‐reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme‐substrate intermediates. The D‐aspartate‐induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.
Kinetic and crystallographic studies of Escherichia coli UDP‐N‐acetylmuramate:L‐alanine ligase
Authors: Emanuele John J. , Jin Haiyong , Jacobson Bruce L. , Chang Chiehying Y. , Einspahr Howard M. , Villafranca Joseph J. 
Published: February 2009

AbstractUridine diphosphate‐N‐acetylmuramate:L‐alanine ligase (EC 6.3.2.8, UNAM:L‐Ala ligase or MurC gene product) catalyzes the ATP‐dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram‐positive and gram‐negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady‐state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH. At 37 °C and pH 8.0, kcat was equal to 980 40 min−1, while Km values for ATP, UNAM, and L‐alanine were, 130 10, 44 3, and 48 6 μM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/Km for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X‐ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 Å, β = 105°, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L‐alanine. This L‐alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L‐alanine. Numerous L‐alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L‐alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L‐alanine analogs tested as inhibitors were competitive versus L‐alanine.
Recombinant human pigment epithelium‐derived factor (PEDF): Characterization of PEDF overexpressed and secreted by eukaryotic cells
Authors: Stratikos Efstratios , Alberdi Elena , Gettins Peter G.W. , Becerra S. Patricia 
Published: February 2009

AbstractPigment epithelium‐derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full‐length glycosylated human recombinant PEDF at high levels (>20 μg/mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full‐length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.
Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance
Authors: Conejero‐Lara Francisco , Parrado Juan , Azuaga Ana I. , Smith Richard A.G. , Ponting Christopher P. , Dobson Christopher M. 
Published: February 2009

AbstractStreptococcus equisimilis streptokinase (SK) is a single‐chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693–704) that SK is composed of three structural domains (A, B, and C) with a C‐terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two‐chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 °C and its stability is largely independent of the presence of the other domains. The high‐temperature transition in intact SK (at approximately 63 °C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63—Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native‐like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native‐like structure of domain A.