bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR
Authors: Huth Jeffrey R. , Bewley Carole A. , Clore G. Marius , Gronenborn Angela M. , Jackson Belinda M. , Hinnebusch Alan G.
Published: February 2009

Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin‐binding domain of streptococcal protein G (GB1 domain) linked to the N‐terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H‐15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15‐labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand‐receptor interactions, using 15N‐labeled GB1‐peptide fusions and unlabeled target.
Characterization of a helix‐loop‐helix (EF hand) motif of silver hake parvalbumin isoform B
Authors: Revett Sandra P. , Nelson Donald J. , King Gina , Shabanowitz Jeffrey , Hunt Donald F. , Laue Thomas M. , Hartman Kari L.
Published: February 2009

Parvalbumins are a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF‐hand. Silver hake parvalbumin (SHPV) consists of three EF‐type helix‐loop‐helix regions, two of which have the ability to bind calcium. The three helix‐loop‐helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV‐B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a β‐lineage parvalbumin. SHPV‐B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33‐amino acid EF fragment (residues 76‐108), containing one of the calcium ion binding sites in native SHPV‐B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion‐induced self‐association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge‐density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer‐dimer equilibrium when complexed with La3+.
Alteration of T4 lysozyme structure by second‐site reversion of deleterious mutations
Authors: Poteete Anthony R. , Rennell Dale , Bouvier Suzanne E. , Hardy Larry W.
Published: February 2009

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second‐site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.
Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry
Authors: Neville David C. A. , Townsend R. Reid , Rozanas Christine R. , Verkman A. S. , Price Elmer M. , Gruis Darren B.
Published: February 2009

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane C1− channel regulated by protein phosphorylation. To identify cAMP‐dependent protein kinase (PKA)‐phosphorylated residues in full‐length CFTR, immobilized metal‐ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron‐loaded (Fe3+) nitrilotriacetic (NTA) Sepharose compared to iminodiacetic acid (IDA) metal‐chelating matrices was demonstrated using a PKA‐phosphorylated recombinant NBD1‐R protein from CFTR. Fe3+‐loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly‐His‐containing peptides were co‐purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA‐phosphorylated full‐length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix‐assisted laser desorption mass spectrometry (MALDI/MS) with post‐source decay (PSD) analysis and collision‐induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full‐length CFTR.
The proteasome 11S regulator subunit REGα (PA28α) is a heptamer
Authors: Johnston Steven C. , Whitby Frank G. , Realini Claudio , Rechsteiner Martin , Hill Christopher P.
Published: February 2009

Activity of the 20s proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REGα and REGβ subunits. We have crystallized recombinant REGα (rREGα) and collected diffraction data to 3.0 Å resolution. The self‐rotation function indicates that rREGα forms a heptameric ring in the crystal. Equilibrium sedimentation demonstrates that rREGα is a heptamer in solution also.
Crystal structures of a marginally active thymidylate synthase mutant, Arg 126 → Glu
Authors: Strop Pavel , Montfort William R. , Changchien Liming , Maley Frank
Published: February 2009

Thymidylate synthase (TS) is a long‐standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co‐enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon‐carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000‐fold and Km reduced 600‐fold. The crystal structure of R126E was determined under two conditions—in the absence of bound ligand (2.4 Å resolution), and with dUMP and the antifolate CB3717 (2.2 Å resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a ∼ 1 Å shift in the phosphate group. Interestingly, the protein displayed the typical ligand‐induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.
A flavodoxin that is required for enzyme activation: The structure of oxidized flavodoxin from Escherichia coli at 1.8 Å resolution
Authors: Hoover David M. , Ludwig Martha L.
Published: February 2009

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate‐lyase, anaerobic ribonucleotide reductase, and B12‐dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P212121, a = 126.4, b = 41.10, c = 69.15 Å, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 Å, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8‐Å resolution were collected at 140 K and the structure was refined to an Rworkof 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non‐hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis‐Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 Å,) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 Å (R = 0.191; Rfree = 0.249). The sequence Tyr 58‐Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.
Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin
Authors: Lazaridis Themis , Lee Irwin , Karplus Martin
Published: February 2009

Molecular dynamics simulations in solution are performed for a rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus (RdPf) and one from the mesophilic organism Desulfovibrio vulgaris (RdDv). The two proteins are simulated at four temperatures: 300 K, 373 K, 473 K (two sets), and 500 K; the various simulations extended from 200 ps to 1,020 ps. At room temperature, the two proteins are stable, remain close to the crystal structure, and exhibit similar dynamic behavior; the RMS residue fluctuations are slightly smaller in the hyperthermophilic protein. An analysis of the average energy contributions in the two proteins is made; the results suggest that the intraprotein energy stabilizes RdPf relative to RdDv. At 373 K, the mesophilic protein unfolds rapidly (it begins to unfold at 300 ps), whereas the hyperthermophilic does not unfold over the simulation of 600 ps. This is in accord with the expected stability of the two proteins. At 473 K, where both proteins are expected to be unstable, unfolding behavior is observed within 200 ps and the mesophilic protein unfolds faster than the hyperthermophilic one. At 500 K, both proteins unfold; the hyperthermophilic protein does so faster than the mesophilic protein. The unfolding behavior for the two proteins is found to be very similar. Although the exact order of events differs from one trajectory to another, both proteins unfold first by opening of the loop region to expose the hydrophobic core. This is followed by unzipping of the β‐sheet. The results obtained in the simulation are discussed in terms of the factors involved in flexibility and thermostability.
Crystallization and preliminary X‐ray diffraction analysis of arginyl‐tRNA synthetase from Escherichia coli
Authors: Lin Sheng‐Xiang , Zhou Ming , Campbell Robert L. , Wang En‐Duo , Wang Ying‐Lai
Published: February 2009

Arginyl‐tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 Å with a rotating anode X‐ray source and R‐axis IIc image plate detector. They have an ortho‐rhombic space group P21212 with unit cell parameters of a = 251.51 Å, b = 53.12 Å, and c = 52.35 Å. A complete native data set has been collected at 3.1 Å resolution for these crystals.
Crystal structure of a non‐toxic mutant of heat‐labile enterotoxin, which is a potent mucosal adjuvant
Authors: Van Den Akker Focco , Pizza Mariagrazia , Rappuoli Rino , Hol Wim G. J.
Published: February 2009

Two closely related bacterial toxins, heat‐labile enterotoxin (LT‐I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single‐site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT‐I is determined at 2.0 Å resolution. Its structure appears to be essentially the same as the wild‐type LT‐I structure. The substitution Ser63Lys was designed, based on the wild‐type LT‐I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT‐I. The fact that the Ser63Lys mutant of LT‐I does not disrupt the wild‐type LT‐I structure makes the non‐toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild‐type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.
Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold
Authors: Buss Kathryn A. , Sanders David A. , Hasson Miriam S. , Ingram‐Smith Cheryl , Ferry James G.
Published: February 2009

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme‐catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 Å resolution. Analysis of X‐ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 Å, b = 67 Å, c = 83 Å, β = 103°. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic twofold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.
Crystallization of the first three domains of the human insulin‐like growth factor‐1 receptor
Authors: Mckern Neil M. , Frenkel Maurice J. , Verkuylen Amanda , Bentley John D. , Lovrecz George O. , Ivancic Neva , Elleman Thomas C. , Cosgrove Leah J. , Lou Meizhen , Garrett THOMAS P. J. , Ward Colin W.
Published: February 2009

The insulin‐like growth factor‐1 receptor (IGF‐1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1‐462) comprising the L1‐cysteine rich‐L2 domains of the human IGF‐1R ectodomain has been overexpressed in glycosylation‐deficient Lec8 cells and has been affinity‐purified via a c‐myc tag followed by gel filtration. The fragment was recognized by two anti‐IGF‐1R monoclonal antibodies, 24‐31 and 24‐60, but showed no detectable binding of IGF‐1 or IGF‐2. Isocratic elution of IGF‐1R/462 on anion‐exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 Å resolution with cell dimensions a = 77.0 A, Å = 99.5 Å, c = 120.1 Å, and space group P212121.
Crystallization and preliminary X‐ray studies of I‐PpoI: A nuclear, intron‐encoded homing endonuclease from Physarum polycephalum
Authors: Flick Karen E. , Mchugh Diane , Stoddard Barry L. , Heath Joe Don , Stephens Kathryn M. , Monnat Raymond J.
Published: February 2009

The homing endonuclease I‐PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26s rRNA gene. This endonuclease promotes the lateral transfer or “homing” of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26s rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I‐PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombi‐nant I‐PpoI has been co‐crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3121, with unit cell dimensions a = b = 114 Å, c = 89 Å. The results of initial X‐ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 Å3/dalton. These experiments also provide strong evidence that I‐PpoI contains several bound zinc ions as part of its structure.
Calculated electrostatic gradients in recombinant human H‐chain ferritin
Authors: Douglas Trevor , Ripoll Daniel R.
Published: February 2009

Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H‐chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three‐fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three‐fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three‐fold channel as the major entrance to the protein.

Pathways from the three‐fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four‐fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 Å), suggesting that electrostatic fields could also play a role in the nucleation process.

A large gradient in the electrostatic potential at the 4‐fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4‐fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de‐mineralization.
Synthesis, physicochemical characterization, and crystallization of a putative retro‐coiled coil
Authors: Liu Niankun , Deillon Christine , Klauser Stephan , Gutte Bernd , Thomas Richard M.
Published: February 2009

An artificial HIV enhancer‐binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Biophys J 25:399‐403). Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct. As the first step toward the synthesis of a basic region‐retro leucine zipper HIV enhancer‐binding fusion protein, we have now prepared the retro version of the leucine zipper (r‐LZ35) and performed initial physicochemical characterization. Circular dichroism and sedimentation equilibrium studies showed that, at concentrations <100 μ, the retro peptide was an unstructured monomer. At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the retro peptide was almost fully helical. N‐terminal extension of the retro peptide by the tripeptide Cys‐Gly‐Gly resulted in a 38‐residue polypeptide that could be covalently dimerized by forming a disulfide bond between two chains to give the peptide (r‐LZ38)2. Even in the low micromolar concentration range peptide (r‐LZ38)2 formed a stable, noncovalent, helical dimer as revealed by circular dichroism and sedimentation equilibrium in the presence and absence of guanidinium chloride. (r‐LZ38)2 has been crystallized and X‐ray structural analysis is under way. The disulfide‐crosslinked retro‐leucine zipper may lend itself to interesting protein structural studies, including protein design. The present work also highlights the structural and functional potential of retro proteins in general.
Assembly and crystallization of the complex between the human T cell coreceptor CD8α homodimer and HLA‐A2
Authors: Gao George F. , Gerth Ulrich C. , Wyer Jessica R. , Willcox Benjamin E. , O'callaghan Christopher A. , Bell John I. , Jakobsen Bent K. , Zhang Zhihong , Jones E. Yvonne
Published: February 2009

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8α was devised using codon usage alterations in the 5′ region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8α, comprising residues 1‐120 of the mature protein, excluding the signal peptide and the membrane‐proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA‐A2 heavy chain, β2‐microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV‐1 were also expressed in E. coli, refolded and purified. CD8α/HLA‐A2 complexes were formed in solution and by co‐crystallization with a stoichiometry of one CD8αα dimer to one HLA‐A2‐peptide unit.
Homologues of 26S proteasome subunits are regulators of transcription and translation
Authors: Aravind L. , Ponting Chris P.
Published: February 2009

Single copies of an α‐helical‐rich motif are demonstrated to be present within subunits of the large multiprotein 26S proteasome and eukaryotic initiation factor‐3 (eIF3) complexes, and within proteins involved in transcriptional regulation. In addition, p40 and p47 subunits of eIF3 are shown to be homologues of the proteasome subunit Mov34, and transcriptional regulators JAB1/pad1. Finally, the proteasome subunit S5a and the p44 subunit of the basal transcription factor IIH (TFIIH) are identified as homologues. The presence of homologous, and sometimes identical, proteins in contrasting functional contexts suggests that the large multisubunit complexes of the 26S proteasome, eIF3 and TFIIH perform overlapping cellular roles.
Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A
Authors: Pearson Matthew A. , Karplus P. Andrew , Dodge Robert W. , Laity John H. , Scheraga Harold A.
Published: February 2009

The Tyr92‐Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide‐intact wild‐type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92‐Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95] A variant, which is an analog of the major rate‐determining three‐disulfide intermediate in the oxidative folding of RNase A, missing the 40‐95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.
Crystallization and preliminary X‐ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H‐D‐Phe‐Phe‐Arg‐chloromethylketone
Authors: Banbula Agnieszka , Potempa Jan , Travis James , Bode Wolfram , Medrano Francisco‐Javier
Published: February 2009

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X‐ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P212121) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 Å resolution, respectively. Cell dimensions are a = 51.9 Å, b = 79.9 Å, and c = 99.6 Å (P212121), and a = b = 176.6 Å, and c = 143.4 Å (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self‐rotation function, could accommodate a tetramer in the asymmetric unit.
Baseline length and automated fitting of denaturation data
Authors: Allen Devon L. , Pielak Gary J.
Published: February 2009

To understand relationships between protein sequence and stability, we often compare data from proteins that differ by the substitution of one amino acid. Frequently, an amino acid change causes the cooperative denaturation transitions to shift to lower temperatures, diminishing the signal from the native state. Here we show that apparent stability changes, i.e., the free energy of denaturation, ΔGD, can also be caused by a deficiency of points in the low temperature end of the transition. In addition, we suggest a method for overcoming this problem.
Structure of malonic acid‐based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data
Authors: Brandstetter Hans , Engh Richard A. , Roedern Erich Graf Von , Moroder Luis , Huber Robert , Bode Wolfram , Grams Frank
Published: February 2009

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid‐based inhibitors using the X‐ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate‐like binding mode. The proposed β‐type turn‐like conformation for the improved inhibitors was confirmed by X‐ray crystallography. The observation of nonsubstrate‐like binding confirms the original strategy for structure‐based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate‐like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500‐fold improved inhibition based on these structures.
Crystal structure of the cyclophilin‐like domain from the parasitic nematode Brugia malayi
Authors: Mikol Vincent , Ma Dong , Carlow Clotilde K.S.
Published: February 2009

Cyclophilins are a family of proteins that exhibit peptidyl‐prolyl cis‐trans isomerase activity and bind the immuno‐suppressive agent Cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin‐like domain was identified at the N‐terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R‐factor of 16.9% at 2.15 Å resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.
Thermodynamic characterization of an intermediate state of human growth hormone
Authors: Gomez‐Orellana Isbuel , Varinano Bruce , Miura‐Fraboni Judy , Milstein Sam , Paton Duncan R.
Published: February 2009

The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two‐state transition was observed between 0 and 100°C. The magnitudes of the ΔH and ΔCP of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n‐propanol, 2‐propanol, or 1,2‐propanediol (propylene glycol), which suggests that the self‐association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7‐8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70‐80% propylene glycol.
Conformational change in the activation of lipase: An analysis in terms of low‐frequency normal modes
Authors: Jääskeläinen Sanna , Verma Chandra S. , Hubbard Roderick E. , Lestko Pekka , Caves Leo S.D.
Published: February 2009

The interfacial activation of Rhizomucor miehei lipase (RmL) involves the motion of an α‐helical region (residues 82‐96) which acts as a “lid” over the active site of the enzyme, undergoing a displacement from a “closed” to an “open” conformation upon binding of substrate. Normal mode analyses performed in both low and high dielectric media reveal that low‐frequency vibrational modes contribute significantly to the conformational transition between the closed and open conformations. In these modes, the lid displacement is coupled to local motions of active site loops as well as global breathing motions. Atomic fluctuations of the first hinge of the lid (residues 83‐84) are substantially larger in the low dielectric medium than in the high dielectric medium. Our results also suggest that electrostatic interactions of Arg86 play an important role in terms of both the intrinsic stability of the lid and its displacement, through enhancement of hinge mobility in a high dielectric medium. Additional calculations demonstrate that the observed patterns of atomic fluctuations are an intrinsic feature of the protein structure and not dependent on the nature of specific energy minima.
Excluded volume in the configurational distribution of a strongly‐denatured protein
Authors: Peterscu Andrei‐Jose , Receveur Veronique , Calmettes Patrick , Durand Dominique , Smith Jeremy C.
Published: February 2009

The configurational distribution of phosphoglycerate kinase (PGK) strongly‐denatured in 4M guanidine hydrochloride solution is investigated using small‐angle neutron scattering (SANS) and Monte Carlo computer simulation. It is shown that the experimental scattering profile can be represented by a random flexible chain of spheres of excess scattering density with excluded volume interactions, the best agreement being achieved when partial sphere intersection is allowed. The radius of gyration of the chain increases by a factor of 4 on denaturation, whereas the average length of segments ˜5 residues long increases by only ˜10%, consistent with a picture in which the large expansion on denaturation originates primarily from increased long‐range flexibility of the polypeptide chain. The results provide a description of the chain statistics from which the construction of starting points for simulation studies of folding of the protein can be envisaged.
From coiled coils to small globular proteins: Design of a native‐like three‐helix bundle
Authors: Bryson James W. , Desjarlais John R. , Handel Tracy M. , Degrado William F.
Published: February 2009

A monomolecular native‐like three‐helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three‐helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine‐induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native‐like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core.
Design, total synthesis, and functional overexpression of the Candida rugosa lipl gene coding for a major industrial lipase
Authors: Brocca Stefania , Schmidt‐Dannert Claudia , Schmid Rolf D. , Lotti Marian , Alberghina Lilia
Published: February 2009

The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip1 gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site‐directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pasto is cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lip1 of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.
Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins
Authors: Whitelegge Julian P. , Gundersen Cameron B. , Faull Kym F.
Published: February 2009

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray‐ionization mass spectrometry (ESI‐MS) of intact intrinsic membrane proteins purified using reverse‐phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the Dl and D2 reaction‐center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI‐MS study of a G‐protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post‐translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI‐MS experiments.
Guanidine hydrochloride unfolding of a transmembrane β‐strand in FepA using site‐directed spin labeling
Authors: Klug Candice S. , Feix Jimmy B.
Published: February 2009

We have used the electron spin resonance (ESR) site‐directed spin‐labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn‐HCl) induced denaturation of several sites along a transmembrane β‐strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the β‐strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027‐13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a β‐barrel motif comprised of a series of antiparallel β‐strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria. We have previously shown that a large surface loop in FepA containing the FeEnt binding site denatures independently of the β‐barrel domain (Klug CS et al., 1995, Biochemistry 34:14230‐14236). The SDSL approach allows examination of the unfolding at individual residues independent of the global unfolding of the protein. This work shows that sites along the β‐strand that are exposed to the aqueous lumen of the channel denature more rapidly and with higher cooperativity than the surface loop, while sites on the hydrophobic side of the β‐strand undergo a limited degree of noncooperative unfolding and do not fully denature even at high (e.g., 4 M) Gdn‐HCl concentrations. We conclude that, in a transmembrane β‐strand, the local environment of a given residue plays a significant role in the loss of structure at each site.
Structure and distribution of pentapeptide repeats in bacteria
Authors: Bateman Alex , Murzin Alexey G. , Teichmann Sarah A.
Published: February 2009

We report the discovery of a novel family of proteins, each member contains tandem pentapeptide (five residue) repeats, described by the motif A(D/N)LXX. Members of this family are both membrane bound and cytoplasmic. The function of these repeats is uncertain, but they may have a targeting or structural function rather than enzymatic activity. This family is most common in cyanobacteria, suggesting a function related to cyanobacterial‐specific metabolism. Although no experimental information is available for the structure of this family, it is predicted that the tandem pentapeptide repeats will form a right‐handed β‐helical structure. A structural model of the pentapeptide repeats is presented.
Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off‐pathway aggregation
Authors: Betts Scott D. , King Jonathan
Published: February 2009

Off‐pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self‐association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off‐pathway aggregation of the parallel β‐coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 °C prior to warming to 20 °C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature‐shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5517‐523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off‐pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450‐455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide‐bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.
MgATP binding to the nucleotide‐binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate‐binding domains
Authors: Szpikowska Barbara K. , Sherman Mark A. , Mas Maria T. , Swiderek Kristine M.
Published: February 2009

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide‐induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of ∼ 30 kDa. Using mass spectrometry (MS) and N‐terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group I1 chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP‐PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group I1 chaperonins may share similar, functionally‐important, conformational changes. Additional conformational changes are likely to involve a flexible helix‐loop‐helix motif, which is characteristic for all group II chaperonins.
Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from antarctic cod
Authors: Lane Andrew N. , Hays Lisa M. , Crowe Lois M. , Crowe John H. , Feeney Robert E.
Published: February 2009

The 1H and 13C NMR spectra of a 14‐residue antifreeze glycopeptide from Antarctic cod (Tetramatornus horchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 °C of 3‐4 ns. The relaxation data and lack of long‐range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16‐residue analogue containing an Arg‐Ala dipeptide added to the C‐terminus. Molecular modeling also showed no evidence of long‐range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or β turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.
Cloning, expression, and purification of a catalytic fragment of moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes
Authors: Sun Dunming , Jessen Sven , Liu Chunhui , Liu Xiuping , Najmudin Shabir , Georgiadis Millie M.
Published: February 2009

Reverse transcriptase is an essential retroviral enzyme that uses RNA‐ and DNA‐directed DNA polymerase activities as well as an RNaseH activity to synthesize a double‐stranded DNA copy of the single‐stranded RNA genome. In an effort to obtain high‐resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment‐nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.