bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity
Authors: Safar Jiri , Roller Peter P. , Gajdusek D. Carleton , Gibbs Clarence J. 
Published: February 2009

AbstractThe scrapie amyloid (prion) protein (PrP27–30) is the protease‐resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of a posttranslational event or conformational abnormality. The conformation, heat stability, and solvent‐induced conformational transitions of PrP27–30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27–30 in films to 60 °C, 100 °C, and 132 °C for 30 min did not change the β‐sheet secondary structure; the infectivity slightly diminished at 132 °C and correlated with a decreased solubility of PrP27–30 in sodium dodecyl sulfate (SDS), probably due to cross‐linking. Exposing PrP27–30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro‐2‐propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent‐exposed and rehydrated solid state PrP27–30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, β‐pleated sheet‐like secondary structure component, an increasing amount of α‐helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the β‐sheet secondary structure of PrP27–30 protein in the solid state. The conformational perturbations of PrP27–30 parallel the changes in infectivity and suggest that the β‐sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.
Structures of randomly generated mutants of T4 lysozyme show that protein stability can be enhanced by relaxation of strain and by improved hydrogen bonding via bound solvent
Authors: Pjura Philip , Matthews Brian W. 
Published: February 2009

AbstractThe structures of three mutants of bacteriophage T4 lysozyme selected using a screen designed to identify thermostable variants are described. Each of the mutants has a substitution involving threonine. Two of the variants, Thr 26 → Ser (T26S) and Thr 151 → Ser (T151S), have increased reversible melting temperatures with respect to the wild‐type protein. The third, Ala 93 → Thr (A93T), has essentially the same stability as wild type. Thr 26 is in the wall of the active‐site cleft. Its replacement with serine results in the rearrangement of nearby residues, most notably Tyr 18, suggesting that the increase in stability may result from the removal of strain. Thr 151 in the wild‐type structure is far from the active site and appears to sterically prevent the access of solvent to a preformed binding site. In the mutant, the removal of the methyl group allows access to the solvent binding site and, in addition, the Ser 151 hydroxyl rotates to a new position so that it also contributes to solvent binding. Residue 93 is in a highly exposed site on the surface of the molecule, and presumably is equally solvent exposed in the unfolded protein. It is, therefore, not surprising that the substitution Ala 93 → Thr does not change stability. The mutant structures show how chemically similar mutations can have different effects on both the structure and stability of the protein, depending on the structural context. The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype. Although knowledge of the mutant structures makes it possible to rationalize their behavior, it would have been very difficult to predict in advance that these mutants would be stabilizing.
Dynamics of interaction of RNA polymerase II with nucleosomes. I. Effect of salts
Authors: Bhargava Purnima 
Published: February 2009

AbstractMononucleosomes were labeled with the sulfhydryl‐specific fluorescence probe 1, 5‐IAEDANS (5‐(2‐((iodoacetyl)amino)ethyl)amino‐naphthalene‐1‐sulfonic acid) by attaching the dye to the single cysteine of H3 through a covalent linkage. The enzyme RNA polymerase II (pol II) utilized the native and the reconstituted fluorescent nucleosomes as templates with greatest efficiency when 0.2 M potassium acetate (AcOK) was used as the supporting salt; 0.2 M NaCl was found to be very much inhibitory. Measurement of polarity of the microenvironment of the dye at its binding site in the nucleosome showed the conformation to be more open in the presence of AcOK, compared to that in 0.1 or 0.2 M NaCl. The binding of pol II to the nucleosome resulted in a relatively more compact structure when measured in terms of the polarity of the microenvironment of the dye in various salt‐dependent conformations of the nucleosomes. Time‐resolved fluorescence spectroscopy showed that the probe molecule at its binding site undergoes certain excited‐state processes, and the presence/absence or rate of these excited‐state processes depends on the conformation of nucleosomes, which in turn depends on the type and concentration of the ion present in the medium. Time‐resolved emission spectra showed that binding of nucleosomes by pol II established some new contacts that resulted in inaccessibility of the dye to the bulk solvent, reflecting a more hydrophobic environment for the dye in the steady‐state spectra. Thus, binding or transcription of nucleosomes by pol II did not break open their structure. Rather, some transient internal adjustments within the histone octamer may take place to accommodate the bulky pol II molecule.
Dynamics of interaction of RNA polymerase II with nucleosomes. II. During read‐through and elongation
Authors: Bhargava Purnima 
Published: February 2009

AbstractThe sulfhydryl‐specific fluorescence probe 1, 5‐IAEDANS (5‐(2‐((iodoacetyl)amino)ethyl)amino‐naphthalene‐1‐sulfonic acid) was attached to the single cysteine of H3, and reconstituted fluorescent mononucleosomes were used as the template for in vitro transcription by the yeast RNA polymerase II (pol II). DNase I digestion analysis revealed that transcription of nucleosomes by pol II resulted in an overall loosening of the structure. Monitoring the transcription event by steady‐state fluorescence analysis showed that nucleosomes only partially open during transcription. This opening is transient in nature, and nucleosomes close back as soon as the pol II falls off the template. Thus, using the technique of fluorescence spectroscopy, partial opening of nucleosome structure could be differentiated from complete dissociation into free DNA and histone octamer, a distinction that may not be possible by techniques like gel electrophoresis. Time‐resolved fluorescence emission spectroscopy suggested that during read‐through of the template by the pol II, histone octamers do not fall off the DNA. Only minor conformational changes within the histone octamer take place to accommodate the transcribing polymerase.
Phosphopeptide binding to the N‐terminal SH2 domain of the p85α subunit of PI 3′‐kinase: A heteronuclear NMR study
Authors: Hensmann Meike , Booker Grant W. , Panayotou George , Boyd Jonathan , Linacre Jeffrey , Waterfield Mike , Campbell Iain D. 
Published: February 2009

AbstractThe N‐terminal src‐homology 2 domain of the p85α subunit of phosphatidylinositol 3′ kinase (SH2‐N) binds specifically to phosphotyrosine‐containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated βPDGF receptor. A titration of a synthetic 12‐residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2‐N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D‐[15N‐1H] heteronuclear single‐quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2‐N domain, these changes indicate a peptide‐binding surface on the protein. Line shape analysis of 1D profiles of individual [15N‐1H]‐HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the [15N‐1H] heteronuclear NOE for the backbone amide of each residue was determined for the SH2‐N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85α SH2‐N domain is suggested by this study.
The tryptophan residues of mitochondrial creatine kinase: Roles of Trp‐223, Trp‐206, and Trp‐264 in active‐site and quaternary structure formation
Authors: Gross Martin , Furter‐Graves Elizabeth M. , Wallimann Theo , Eppenberger Hans M. , Furter Rolf 
Published: February 2009

AbstractThe 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib‐CK) were individually replaced by phenylalanine or cysteine using site‐directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp‐223 is identified as an active‐site residue whose replacement even by phenylalanine resulted in ≥96% inactivation of the enzyme. Trp‐223 is responsible for a strong (18–21%) fluorescence quenching effect occurring upon formation of a transition state–analogue complex (TSAC; Mib‐CK·creatine·MgADP·NO3–), and Trp‐223 is probably required for the conformational change leading to the TSAC‐induced octamer dissociation of Mib‐CK. Replacement of Trp‐206 by cysteine led to a destabilization of the active‐site structure, solvent exposure of Trp‐223, and to the dissociation of the Mib‐CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp‐264 is shown to be located at the dimer–dimer interfaces within the Mib‐CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC‐induced octamer dissociation. Substitution of Trp‐264 by cysteine drastically accelerated the TSAC‐induced dissociation and destabilized the octameric structure by one‐fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp‐213 and Trp‐268, could be less well assigned.
Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain 6‐N‐acetyllysine
Authors: Violand Bernard N. , Schlittler Michael R. , Lawson Cory Q. , Kane James F. , Siegel Ned R. , Smith Christine E. , Kolodziej Eric W. , Duffin Kevin L. 
Published: February 2009

AbstractRecombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, ←‐N‐acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed‐phase HPLC (RP‐HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP‐HPLC purified protein were used to identify this altered residue as ←‐N‐acetyllysine. Ion‐exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in ←‐N‐acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion‐exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG‐purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of ←‐N‐acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.
Proteolytic excision and in situ cyclization of a bioactive loop from an REI‐VL presentation scaffold
Authors: Helms Larry R. , Wetzel Ronald 
Published: February 2009

AbstractCovalent cyclization of peptides is an important tool in structure–function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor‐bound conformation. Previously we described a 2‐step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N‐terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533–539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo‐LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI‐VL) with an RGD‐containing sequence engineered into its CDR3 and flanked by Lys residues. This REI‐VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor αIIbβ3. The RGD‐containing peptide excised by endo‐LysC from the REI‐VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, an IC50 of about 10 nM. Significantly, both the Nα‐acylation and the cyclization reactions occur efficiently even in the context of the other endo‐LysC fragments of REI‐VL, which suggests that the reaction may prove useful in converting mixtures of endo‐LysC products of many proteins into the corresponding cyclic peptides in situ.
Expression, purification, and characterization of thymidylate synthase from Lactococcus lactis
Authors: Greene Patricia J. , Yu Pak‐Lam , Zhao Jia , Schiffer Celia A. , Santi Daniel 
Published: February 2009

AbstractThe thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q‐Sepharose and phenyl‐Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3‐dimensional homology model, we have predicted covariant changes that might compensate for these differences. With the large amounts of L. lactis TS now available, studies can be pursued to understand the structure‐function relationships of this enzyme compared to other TSs and to confirm the presumed roles of the compensatory changes predicted in the homology model.
Structural similarity between binding sites in influenza sialidase and isocitrate dehydrogenase: Implications for an alternative approach to rational drug design
Authors: Poirrette Andrew R. , Grindley Helen M. , Rice David W. , Artymiuk Peter J. , Willett Peter 
Published: February 2009

AbstractUsing searching techniques based on algorithms derived from graph theory, we have established a similarity between a 3‐dimensional cluster of side chains implicated in drug binding in influenza sialidase and side chains involved in isocitrate binding in Escherichia coli isocitrate dehydrogenase. The possible implications of the use of such comparative methods in drug design are discussed.
Transglutaminase factor XIII uses proteinase‐like catalytic triad to crosslink macromolecules
Authors: Pedersen Lars C. , Yee Vivien C. , Bishop Paul D. , Trong Isolde Le , Teller David C. , Stenkamp Ronald E. 
Published: February 2009

AbstractThe X‐ray crystal structure of human transglutaminase factor XIII has revealed a cysteine proteinase‐like active site involved in a crosslinking reaction and not proteolysis. This is among the first observations of similar active sites in 2 different enzyme families catalyzing a similar reaction in opposite directions. Although the size and overall protein fold of factor XIII and the cysteine proteinases are quite different, the active site and the surrounding protein structure share structural features suggesting a common evolutionary lineage. Here we present a description of the residues in the active site and the structural evidence that the catalytic mechanism of the transglutaminases is similar to the reverse mechanism of the cysteine proteinases.
Collectins — soluble proteins containing collagenous regions and lectin domains — and their roles in innate immunity
Authors: Hoppe Hans‐Jürgen , Reid Kenneth B.M. 
Published: February 2009

AbstractThe collectins are a group of mammalian lectins containing collagen‐like regions. They include mannan binding protein, bovine conglutinin, lung surfactant protein A, lung surfactant protein D, and a newly discovered bovine protein named collectin‐43. These proteins share a very similar modular domain composition and overall 3‐dimensional structure. They also appear to play similar biological roles in the preimmune defense against microorganisms in both serum and lung surfactant. The close evolutionary relationship between the collectins is further emphasized by a common pattern of exons in their genomic structures and the presence of a gene cluster on chromosome 10 in humans that contains the genes known for the human collectins. Studies on the structure/function relationships within the collectins could provide insight into the properties of a growing number of proteins also containing collagenous regions such as Clq, the hibernation protein, the α‐ and β‐ficolins, as well as the membrane acetylcholinesterase and the macrophage scavenger receptor.
Primary structure and functional characterization of rat C5a: An anaphylatoxin with unusually high potency
Authors: Cui Lianxian , Carney David F. , Hugli Tony E. 
Published: February 2009

AbstractThe anaphylatoxin C5a is a pro‐inflammatory factor generated from C5 during complement activation. C5a derived from rat C5 exhibits significantly greater potency compared to C5a from other species. Rat C5a was 25‐fold more potent than human C5a for eliciting spasmogenic contraction of guinea pig ileum. Proteolytic removal of the C‐terminal arginine of C5a (C5adesArg) reduced spasmogenic potency of rat C5a by only 4‐fold compared to a 3,000‐fold reduction for human C5adesArg. In addition, rat C5adesArg was 50‐fold more potent than human C5adesArg in a guinea pig vascular permeability (in vivo) assay and as a chemotactic factor for human neutrophils. C5a and C5adesArg were purified from zymosan‐activated rat serum. Rat C5a, like human C5a, is glycosylated but contains 77 amino acid residues instead of the 74 residues of human C5a. Comparison of the primary structures of rat and human C5a indicated differences at 30 positions including an insert of 3 residues (LLH) in the rat molecule between residue positions 3 and 4 in human C5a. Insertion of residues LLH between Gln‐3 and Lys‐4 in a recombinant human C5a molecule using site‐directed mutagenesis failed to enhance potency. Synthetic C‐terminal analogues of rat C5a proved to be measurably more potent than the corresponding human C5a analogues (Ember JA et al., 1993, Protein Sci 2(Suppl 1): 159 [Abstr]). We conclude that multiple sequence differences in the C‐terminal effector portion and/or elsewhere in rat C5a, but not the LLH insert, account for the significant enhancement in potency of rat C5a over C5a from other species.
A survey of furin substrate specificity using substrate phage display
Authors: Matthews David J. , Goodman Laurie J. , Gorman Cornelia M. , Wells James A. 
Published: February 2009

AbstractThe substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate‐alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7‐residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.
Buried waters and internal cavities in monomeric proteins
Authors: Williams Mark A. , Goodfellow Julia M. , Thornton Janet M. 
Published: February 2009

AbstractWe have analyzed the buried water molecules and internal cavities in a set of 75 high‐resolution, nonhomologous, monomeric protein structures. The number of hydrogen bonds formed between each water molecule and the protein varies from 0 to 4, with 3 being most common. Nearly half of the water molecules are found in pairs or larger clusters. Approximately 90% are shown to be associated with large cavities within the protein, as determined by a novel program, PRO_ACT. The total volume of a protein's large cavities is proportional to its molecular weight and is not dependent on structural class. The largest cavities in proteins are generally elongated rather than globular. There are many more empty cavities than hydrated cavities. The likelihood of a cavity being occupied by a water molecule increases with cavity size and the number of available hydrogen bond partners, with each additional partner typically stabilizing the occupied state by 0.6 kcal/mol.
Ligation alters the pathway of urea‐induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase
Authors: Bromberg S. , Licata V.J. , Mallikarachchi D. , Allewell N.M. 
Published: February 2009

AbstractWe have examined the pathway and energetics of urea‐induced dissociation and unfolding of the catalytic trimer (C3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N‐(phosphonacetyl)‐L‐Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl− and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active‐site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for ΔGdissociation at 7.5 °C of about 7–10 kcal m−1 per interface. ΔGunfolding of dissociated chains when modeled as a 2‐state process is estimated to be very small, on the order of ˜2 kcal m−1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1‐state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl−, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.