bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin
Authors: Correll Carl C. , Ludwig Martha L. , Bruns Christopher M. , Karplus P. Andrew
Published: February 2009

The structure of phthalate dioxygenase reductase (PDR), a monomeric iron‐sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin‐NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe‐2S] are very similar in the two systems. Alignment of the X‐ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P‐450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel β‐barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide‐binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753–3759).

Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2′ phosphate of NADP+. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN.

The midpoint potentials for reduction of the flavin and [2Fe–2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.
Modeling the αIIbβ3 integrin solution conformation
Authors: Rocco Mattia , Spotorno Bruno , Hantgan Roy R.
Published: February 2009

The αIIbβ3 platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of α and β subunits. Electron microscopic (EM) images of the αIIbβ3 complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross‐linking studies rather suggest a somewhat more compact conformation for the αIIbβ3 complex.

We have studied the shape of detergent‐solubilized αIIbβ3 by employing a low‐resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three‐dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and ‐18% from the experimental values, determined, respectively, in octyl glucoside and Triton X‐100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by ‐7% (octyl glucoside) to ‐6% (Triton X‐100).

Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent‐solubilized αIIbβ3 complex, where the relative spatial location of specific domains the size of 5–10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.
Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease
Authors: Tomasselli Alfredo G. , Sarcich Jean L. , Barrett Linda J. , Reardon Ilene M. , Howe W. Jeffrey , Evans David B. , Sharma Satish K. , Heinrikson Robert L.
Published: February 2009

A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV‐1) to digestion by the HIV‐1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440‐Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227–14232). The COOH‐terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483‐Leu484 and Tyr532. Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436‐Ala437 and at Phe440‐Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.
P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences
Authors: Milla Marcos E. , Brown Bronwen M. , Sauer Robert T.
Published: February 2009

Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C‐terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10‐ to 20‐fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, HHHHHH (st6), was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild‐type Arc. The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel‐chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background. The thermodynamic stability of the EA28 mutant (ΔΔGu ‐0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (ΔΔGu ‐3.0 kcal/mol) and SA32 mutant (ΔΔGu ‐3.3 kcal/mol) are substantially less stable.
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

The 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

The 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

Mononucleosomes 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

The 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

The 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

The 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

Recombinant 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

Covalent 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

The 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

Using 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

The 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

The 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

The 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

The 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

We 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

We 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.
Stability of yeast iso‐1‐ferricytochrome c as a function of pH and temperature
Authors: Cohen David S. , Pielak Gary J.
Published: February 2009

Absorbance‐detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso‐1‐ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2‐state model. Values for free energy (ΔGD), enthalpy (ΔHD), and entropy (ΔSD) of denaturation were determined as functions of pH and temperature. The value of ΔGD at 300 K, pH 4.6, is 5.1 ± 0.3 kcal mol−1. The change in molar heat capacity upon denaturation (ΔCp), determined by the temperature dependence of ΔHD as a function of pH (1.37 ± 0.06 kcal mol−1 K−1), agrees with the value determined by differential scanning calorimetry. pH‐dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a ΔHD of 1.30 kcal mol−1 residue−1 at 373.6 K and a ΔSD of 4.24 cal mol−1 K−1 residue−1 at 385.2 K. These values agree with those for other single‐domain, globular proteins.
Determination of the structure of the DNA binding domain of γΔ resolvase in solution
Authors: Liu Tun , Derose Eugene F. , Mullen Gregory P.
Published: February 2009

The DNA binding domain (DBD) of γΔ resolvase (residues 141–183) is responsible for the interaction of this site‐specific DNA recombinase with consensus site DNA within the γΔ transposable element in Escherichia coli. Based on chemical‐shift comparisons, the proteolytically isolated DBD displays side‐chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309–16315). The structure of the DBD in solution has been determined using restraints obtained from 2‐dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 ϕ and X1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data. With respect to the computed average structure, these conformers display an RMS deviation of 0.67 Å for the heavy backbone atoms and 1.49 Å for all heavy atoms within residues 149–180. The DBD consists of 3 α‐helices comprising residues D149–Q157, S162–T167, and R172–N183. Helix‐2 and helix‐3 form a backbone fold, which is similar to the canonical helix‐turn‐helix motif. The conformation of the NH2‐terminal residues, G141–R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix‐1 and helix‐3 cross with a right‐handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix‐3 in the major groove and the amino‐terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by γΔ resolvase and provides initial structure‐function comparisons among the divergent DBDs of related resolvases and invertases.
Deuterium exchange of α‐helices and β‐sheets as monitored by electrospray ionization mass spectrometry
Authors: Wagner David S. , Melton Laura G. , Yan Yibing , Erickson Bruce W. , Anderegg Robert J.
Published: February 2009

Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI‐MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several α‐helical peptides and β‐sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, α‐helical, or β‐sheet structure. For 3 isomeric 31‐residue α‐helical peptides, the number of slowly exchanging hydrogens as measured by ESI‐MS in 50% CF3CD2OD (pD 9.5) provided estimates of their α‐helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of β‐sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD3OD/0.5% CD3CO2D (pD 3.8) as measured by ESI‐MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI‐MS requires much less sample (1–50 μg), much shorter analysis time (10–90 min), and no chemical quenching of the exchange reaction.
Recognition of related proteins by iterative template refinement (ITR)
Authors: Yi Tau‐Mu , Lander Eric S.
Published: February 2009

Predicting the structural fold of a protein is an important and challenging problem. Available computer programs for determining whether a protein sequence is compatible with a known 3‐dimensional structure fall into 2 categories: (1) structure‐based methods, in which structural features such as local conformation and solvent accessibility are encoded in a template, and (2) sequence‐based methods, in which aligned sequences of a set of related proteins are encoded in a template. In both cases, the programs use a static template based on a predetermined set of proteins. Here, we describe a computer‐based method, called iterative template refinement (ITR), that uses templates combining structure‐based and sequence‐based information and employs an iterative search procedure to detect related proteins and sequentially add them to the templates. Starting from a single protein of known structure, ITR performs sequential cycles of database search to construct an expanding tree of templates with the aim of identifying subtle relationships among proteins. Evaluating the performance of ITR on 6 proteins, we found that the method automatically identified a variety of subtle structural similarities to other proteins. For example, the method identified structural similarity between arabinose‐binding protein and phosphofructokinase, a relationship that has not been widely recognized.
A new family of carbon‐nitrogen hydrolases
Authors: Bork Peer , Koonin Eugene V.
Published: February 2009

Using computer methods for database search and multiple alignment, statistically significant sequence similarities were identified between several nitrilases with distinct substrate specificity, cyanide hydratases, aliphatic amidases, β‐alanine synthase, and a few other proteins with unknown molecular function. All these proteins appear to be involved in the reduction of organic nitrogen compounds and ammonia production. Sequence conservation over the entire length, as well as the similarity in the reactions catalyzed by the known enzymes in this family, points to a common catalytic mechanism. The new family of enzymes is characterized by several conserved motifs, one of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis.
Protein identification in DNA databases by peptide mass fingerprinting
Authors: James Peter , Quadroni Manfredo , Carafoli Ernesto , Gonnet Gaston
Published: February 2009

Proteins can be identified using a set of peptide fragment weights produced by a specific digestion to search a protein database in which sequences have been replaced by fragment weights calculated for various cleavage methods. We present a method using multidimensional searches that greatly increases the confidence level for identification, allowing DNA sequence databases to be examined. This method provides a link between 2‐dimensional gel electrophoresis protein databases and genome sequencing projects. Moreover, the increased confidence level allows unknown proteins to be matched to expressed sequence tags, potentially eliminating the need to obtain sequence information for cloning. Database searching from a mass profile is offered as a free service by an automatic server at the ETH, Zürich. For information, send an electronic message to the address cbrg@inf.ethz.ch with the line: help mass search, or help all.
Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone
Authors: Shastry M. C. R. , Agashe Vishwas R. , Udgaonkar Jayant B.
Published: February 2009

The fluorescence‐monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 °C, have been quantitatively analyzed using a 3‐state mechanism: US → UF →N. US and UF are slow‐refolding and fast‐refolding unfolded forms of barstar, and N is the native protein. US and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47‐Pro 48 bond. The 3‐state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3‐state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio US:UF (K21) and the values of the rate constants of the US → UF and UF → US reactions, k12 and k21 respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 ± 0.1, and k12 and k21 have values of 5.3 × 10−3 s−1 and 11.2 × 10−3 s−1, respectively. Double‐jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3‐state analysis and thereby the validity of the 3‐state model. The 3‐state model does not account for the kinetics of folding in the pretransition region, where folding occurs by 2 parallel pathways, UF → N, and US → IN → N, and IN is a native‐like intermediate. The rate constants of the UF → N and US → IN reactions are both similar, with values of 37 s−1 in water. The IN → N reaction, which involves the same trans‐cis isomerization process as the US → UF reaction, occurs with a rate constant of 16 × 10−3 s−1 and is independent of GdnHCl concentration. Thus, trans‐cis isomerization occurs 3 times faster in the folding intermediate than in the unfolded state.
Refined structure of dimeric diphtheria toxin at 2.0 Å resolution
Authors: Bennett M. J. , Choe S. , Eisenberg David
Published: February 2009

The refined structure of dimeric diphtheria toxin (DT) at 2.0 Å resolution, based on 37,727 unique reflections (F > 1σ(F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3′‐5′ uridine 3′ monophosphate (ApUp), and 405 well‐ordered water molecules. The 2.0‐Å refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor‐binding domains and is different from the Rossmann dinucleotide‐binding fold. ApUp is bound in part by a long loop (residues 34–52) that crosses the active site. Several residues in the active site were previously identified as NAD‐binding residues. Glu 148, previously identified as playing a catalytic role in ADP‐ribosylation of elongation factor 2 by DT, is about 5 Å from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an “open” structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by “domain swapping” to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.
Refined structure of monomelic diphtheria toxin at 2.3 Å resolution
Authors: Bennett M. J. , Eisenberg David
Published: February 2009

The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 Å resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3′‐5′ uridine 3′ monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the “open” monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000–0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main‐chain conformations of 8 residues acting as a hinge to “open” or “close” the receptor‐binding (R) domain, and (2) a possible receptor‐docking site, a β‐hairpin loop protruding from the R domain containing residues that bind the cell‐surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5‐step structure‐based mechanism of intoxication: (1) proteolysis of a disulfide‐linked surface loop (residues 186–201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a β‐hairpin loop protruding from the R domain to the DT receptor, leading to receptor‐mediated endocytosis; (3) low pH‐triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP‐ribosylation of elongation factor 2.
Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor
Authors: Orlandini Maurizio , Santucci Annalisa , Tramontano Anna , Neri Paolo , Oliviero Salvatore
Published: February 2009

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3‐dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody‐Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site‐directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.