bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of phlorizin, a potent inhibitor of the Na+/D‐glucose cotransporter, with the NADPH‐binding site of mammalian catalases
Authors: Kitlar Tomas , Döring Frank , Kinne Rolf K.H. , Deutscher Josef , Diedrich Donald F. , Frank Rainer , Wallmeier Holger
Published: July 2010

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D‐glucose cotransporter. In an attempt to purify the Na+/D‐glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi‐Gel affinity chromatography column to which 3‐aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crussa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 μM NADPH from phlorizin columns. Irradiation in the presence of [3H]4‐azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 μM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.
A structural basis for the interaction of urea with lysozyme
Authors: Pike Ashley C.W. , Acharya K. Ravi
Published: July 2010

The effect of urea on the crystal structure of hen eggwhite lysozyme has been investigated using X‐ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type I1 crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 Å resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 Å. Conformational changes are limited to flexible surface loops (Thr 69‐Asn 74, Ser 100‐Asn 103), the active site loop (Asn 59‐Cys go), and the C‐terminus (Cys 127‐Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N‐acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.
A novel zinc‐binding motif found in two ubiquitous deaminase families
Authors: Reizer Jonathan , Buskirk Stewart , Reizer Aiala , Saier Milton H. , Bairoch Amos
Published: July 2010

Two families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc‐binding motif, here designated ZBS. We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc‐binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available.
Thermodynamics of staphylococcal nuclease denaturation. II. The A‐state
Authors: Carra John H. , Anderson Elizabeth A. , Privalov Peter L.
Published: July 2010

Staphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded or molten globule form called the “A‐state” (Fink et al., 1993, Protein Sci 2:1155–1160). We have found that the A‐state of nuclease at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A‐state has the far‐UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A‐state denatures with a sigmoidal change in far‐UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A‐state in the same way as they affect the denaturation of the native state. The A‐state must, therefore, contain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A‐state is most stable at temperatures below 0°C.
Crystallization and preliminary structural studies of neurotrophin‐3
Authors: Kelly Jennifer A. , Singer Elizabeth , Osslund Timothy D. , Yeates Todd O.
Published: July 2010

Neurotrophin‐3 (NT‐3) has been crystallized in 2 forms. Orthorhombic crystals, space group P21212, diffracted to 2.8 Å and have cell dimensions a = 39.1 Å, b = 54.0 Å, and c = 65.5 Å. The second form is space group P43212, with cell dimensions a = b = 67.1 Å, and c = 107.9 Å. The tetragonal crystals diffract to 2.8 Å at room temperature and 2.5 Å at −100 °C. The unit cell dimensions change significantly upon freezing, a = b = 66.1 Å, and c = 102.8 Å. Phases for the orthorhombic form were obtained by molecular replacement using nerve growth factor as the search model. A partially refined model of the NT‐3 dimer (75% complete) was then oriented and positioned in the tetragonal cell.
Conformational stability of HPr: The histidine‐containing phosphocarrier protein from Bacillus subtilis
Authors: Martin Scholtz J.
Published: July 2010

The conformational stability of the histidine‐containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea‐induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (ΔHg, Tg, ΔSg, and ΔCp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520–2525) was used to estimate ΔCP as was a global analysis of the thermal‐ and urea‐induced unfolding data. Each method used to analyze the data gives a similar value for ΔCp (1,170 Δ 50 cal mol−1 K−1). Despite the high melting temperature for HPr (Tg = 73.5 °C), the maximum stability of the protein, which occurs at 26 °C, is quite modest (AGs = 4.2 kcal mol−1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of ΔCp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685–691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.
Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase
Authors: Egloff Marie‐Pierre , Sarda Louis , Verger Robert , Cambillau Christian , Tilbeurgh Herman Van
Published: July 2010

Colipase (Mr 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt concentrations). Previously determined 3‐Å‐resolution X‐ray structures of lipase‐colipase complexes have shown that, in the absence of substrate, colipase binds to the noncatalytic C‐terminal domain of pancreatic lipase (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 559:159–162; van Tilbeurgh et al., 1993a, Nature 362:814–820). Upon lipid binding, conformational changes at the active site of pancreatic lipase bring a surface loop (the lid) in contact with colipase, creating a second binding site for this cofactor. Covalent inhibition of the pancreatic lipase by a phosphonate inhibitor yields better diffracting crystals of the lipase‐colipase complex. From the 2.4‐Å‐resolution structure of this complex, we give an accurate description of the colipase. It confirms the previous proposed disulfide connections (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159–162; van Tilbeurgh et al., 1993a, Nature 362:814–820) that were in disagreement with the biochemical assignment (Chaillan C, Kerfelec B, Foglizzo E, Chapus C, 1992, Biochem Biophys Res Commun 184:206–211). Colipase lacks well‐defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger‐like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C‐terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.
Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three‐dimensional model for a core region in protein phosphatases
Authors: Eckstein Jens W. , Beer‐Romero Peggy , Berdo Ingrid
Published: July 2010

The reaction mechanism of protein tyrosine phosphatases (PTPases) and dual‐specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site‐directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site‐directed mutagenesis. The D383N mutant enzyme exhibits a 150‐fold reduction in kcat, with Km only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two‐domain structure for Cdc25, with a regulatory N‐terminal and a catalytic C‐terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three‐dimensional model for the core region of Cdc25. By comparing this three‐dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.
Structure in solution of a four‐helix lipid binding protein
Authors: Heinemann Bo , Andersen Kim Vilbour , Nielsen Peter Reinholt , Bech Lene Mølskov , Poulsen Flemming M.
Published: July 2010

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three‐dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91‐residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H‐1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 ø‐angles, 61 χ1 angles, and 2 χ2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well‐defined α‐helices A‐D (A, Cys 3‐Gly 19; B, Gly 25‐Ala 38; C, Arg 44‐Gly 57; D, Leu 63‐Cys 73), separated by three short loops that are less well defined and concluded by a well defined C‐terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 Å 0.04 Å for backbone atoms and 0.93 Å± 0.06 Å for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413–422; Shin DH et al, 1995, Structure 3:189–199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.
Interaction of partially unfolded forms ofTorpedoacetylcholinesterase with liposomes
Authors: Shin Irina , Silman Israel , Weiner Lev M.
Published: July 2010

A water‐soluble dimeric form of acetylcholinesterase from electric organ tissue ofTorpedo californicawas obtained by solubilization with phosphatidylinositol‐specific phospholipase C of the glycophosphatidylinositol‐anchored species, followed by purification by affinity chromatography. The water‐soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head‐group region of the bilayer. The effects of tryptic digestion of the bound acetylcholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass ∼5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268‐Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274‐Met 308, as identified on the basis of hydropathy plots. Inspection of the three‐dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
A structural basis for enantioselective inhibition of Candida rugosa lipase by long‐chain aliphatic alcohols
Authors: Holmquist Mats , Hæffner Fredrik , Norin Torbjörn , Hult Karl
Published: July 2010

Molecular modeling showed that the enantiomers of heptyl 2‐methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast‐reacting S‐enantiomer may well occupy the previously identified acyl‐binding tunnel in the active site of the lipase. By contrast, the slow‐reacting R‐enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long‐chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2‐methyldecanoic acid. Long‐chain aliphatic alcohols may coordinate to the acyl‐binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast‐reacting S‐enantiomer, thus resulting in a lowered enantioselectivity in the resolution.
Improving protein secondary structure prediction with aligned homologous sequences
Authors: Di Francesco V. , Garnier J. , Munson P.J.
Published: July 2010

Most recent protein secondary structure prediction methods use sequence alignments to improve the prediction quality. We investigate the relationship between the location of secondary structural elements, gaps, and variable residue positions in multiple sequence alignments. We further investigate how these relationships compare with those found in structurally aligned protein families. We show how such associations may be used to improve the quality of prediction of the secondary structure elements, using the Quadratic‐Logistic method with profiles. Furthermore, we analyze the extent to which the number of homologous sequences influences the quality of prediction. The analysis of variable residue positions shows that surprisingly, helical regions exhibit greater variability than do coil regions, which are generally thought to be the most common secondary structure elements in loops. However, the correlation between variability and the presence of helices does not significantly improve prediction quality. Gaps are a distinct signal for coil regions. Increasing the coil propensity for those residues occurring in gap regions enhances the overall prediction quality. Prediction accuracy increases initially with the number of homologues, but changes negligibly as the number of homologues exceeds about 14. The alignment quality affects the prediction more than other factors, hence a careful selection and alignment of even a small number of homologues can lead to significant improvements in prediction accuracy.
Solvent isotope effects on the refolding kinetics of hen egg‐white lysozyme
Authors: Itzhaki Laura S. , Evans Philip A.
Published: July 2010

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate‐determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.
The protein machinery of vesicle budding and fusion
Authors: Rothman James E.
Published: July 2010

A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry.
Crystallographic determination of the structures of human α‐thrombin complexed with BMS‐186282 and BMS‐189090
Authors: Malley Mary F. , Sack John S. , Tabernero Lydia , Chang Chiehying Y. , Ohringer Shari L. , Roberts Daniel G. M. , Das Jagabandhu
Published: July 2010

The crystallographic structures of the ternary complexes of human α‐thrombin with hirugen (a sulfated hirudin fragment) and the small‐molecule active site thrombin inhibitors BMS‐186282 and BMS‐189090 have been determined at 2.6 and 2.8 Å. In both cases, the inhibitors, which adopt very‐similar bound conformations, bind in an antiparallel β‐strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D‐Phe‐Pro‐Arg‐CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS‐186282 nor BMS‐189090 bind covalently and only BMS‐186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.
Folding proteins with a simple energy function and extensive conformational searching
Authors: Yue Kaizhi , Dill Ken A.
Published: July 2010

We describe a computer algorithm for predicting the three‐dimensional structures of proteins using only their amino acid sequences. The method differs from others in two ways: (1) it uses very few energy parameters, representing hydrophobic and polar interactions, and (2) it uses a new “constraint‐based exhaustive” searching method, which appears to be among the fastest and most complete search methods yet available for realistic protein models. It finds a relatively small number of low‐energy conformations, among which are native‐like conformations, for cram‐bin (1CRN), avian pancreatic polypeptide (1PPT), melittin (2MLT), and apamin. Thus, the lowest‐energy states of very simple energy functions may predict the native structures of globular proteins.
Yeast heat shock transcription factor N‐terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy
Authors: Cho Ho S. , Liu Corey W. , Damberger Fred F. , Pelton Jeffrey G. , Nelson Hillary C. M. , Wemmer David E.
Published: July 2010

The structure and dynamics of the N‐terminal activation domains of the yeast heat shock transcription factors of Kluyveromyces lactis and Saccharomyces cerevisiae were probed by heteronuclear 15N[1H] correlation and 15N[1H] NOE NMR studies. Using the DNA‐binding domain as a structural reference, we show that the protein backbone of the N‐terminal activation domain undergoes rapid, large‐amplitude motions and is therefore unstructured. Difference CD data also show that the N‐terminal activation domain remains random‐coil, even in the presence of DNA. Implications for a “polypeptide lasso” model of transcriptional activation are discussed.
Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase
Authors: Agarwalla Sanjay , Gokhale Rajesh S. , Balaram P. , Santi Daniel V.
Published: July 2010

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3–5 M), well below that required for complete denaturation, as established by fluorescence and size‐exclusion chromatography. In contrast to the wild‐type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155–188′ and 188–155′, does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non‐native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein‐protein interfaces may be a useful strategy in minimizing aggregation of non‐native structures in multimeric proteins.
Structural coupling of the inhibitory regions flanking the ETS domain of murine Ets‐1
Authors: Donaldson Logan W. , Mcintosh Lawrence P. , Skalicky Jack J. , Petersen Jeannine M. , Graves Barbara J.
Published: July 2010

Several members of the ets gene family of transcription factors show negative regulation of DNA binding by intramolecular interactions. A structural mechanism for this auto‐inhibition is investigated using a 161‐residue N‐terminal deletion mutant of murine Ets‐1, Ets‐1ΔN280. This protein shows a similar reduced affinity for DNA as native Ets‐1 because it contains the ETS domain in context of the flanking amino‐ and carboxy‐terminal regions that together mediate repression of DNA binding. The secondary structure of Ets‐1ΔN280 was determined using NMR chemical shift, NOE, J coupling, and amide hydrogen exchange information. In addition to the winged helix‐turn‐helix ETS domain, Ets‐1ΔN280 contains two α‐helices in the amino‐terminal inhibitory region and one α‐helix in the carboxy‐terminal inhibitory region. Chemical shift comparisons were made between this protein and an activated form of Ets‐1 lacking the amino‐terminal inhibitory region. The spectral differences demonstrate that the amino‐ and carboxy‐terminal inhibitory sequences are structurally coupled to one another, thus explaining the observation that both regions are required for the repression of DNA binding. Furthermore, these data show that the inhibitory sequences also interact directly with the first helix of the intervening ETS domain, thereby providing a pathway for the repression of DNA binding. These results lead to a model of an inhibitory module in Ets‐1 composed of both the amino‐ and carboxy‐terminal regions interfaced with the ETS domain. This establishes the structural framework for understanding the intramolecular inhibition of Ets‐1 DNA binding.
Topology prediction of membrane proteins
Authors: Argos Patrick , Persson Bengt
Published: July 2010

A new method is described for prediction of protein membrane topology (intra‐ and extracellular sidedness) from multiply aligned amino acid sequences after determination of the membrane‐spanning segments. The prediction technique relies on residue compositional differences in the protein segments exposed at each side of the membrane. Intra/extracellular ratios are calculated for the residue types Asn, Asp, Gly, Phe, Pro, Trp, Tyr, and Val, preferably found on the extracellular side, and for Ala, Arg, Cys, and Lys, mostly occurring on the intracellular side. The consensus over these 12 residue distributions is used for sidedness prediction. The method was developed with a test set of 42 protein families, for which all but one were correctly predicted with the new algorithm. This represents an improvement over predictions based on the widely used “positive‐inside rule” and other techniques, where at least six mispredictions were observed for the same data set. Further, application of this and other methods to 12 protein families not in the test set still showed the better performance of the present technique, which was subsequently applied to another set of membrane protein families where the topology has yet to be determined.
Crystallization of a soluble form of the Kexlp serine carboxypeptidase from Saccharomyces cerevisiae
Authors: Tessier Daniel , Thomas David Y. , Shilton Brian H. , Li Yunge , Cygler Miroslaw
Published: July 2010

A soluble form of the killer factor and prohormone‐processing carboxypeptidase, “KexlΔp,” from Saccharomyces cerevisiae, has been crystallized in 17–22% poly (ethylene glycol) methyl ether (average Mr = 5, 000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 °C. A native data set (2.8 Å resolution) and four derivative data sets (3.0–3.2 Å resolution) were collected at the Photon Factory (λ = 1.0 Å). The crystals belong to space group P212121 with a = 56.6 Å, b = 84.0 Å, c= 111.8 Å. Freezing a KexlΔp crystal has facilitated the collection of a 2.4‐Å data set using a rotating anode source (λ = 1.5418 Å). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW‐II; Liao DI et al., 1992, Biochemistry 31:9796–9812) and yeast carboxypeptidase Y (CPD‐Y; Endrizzi JA, Breddam K, Remington SJ, 1994, Biochemistry 33:11106–11120).
Overexpression of bacterio‐opsin in Escherichia coli as a water‐soluble fusion to maltose binding protein: Efficient regeneration of the fusion protein and selective cleavage with trypsin
Authors: Chen Guo‐Qiang , Gouaux J. Eric
Published: July 2010

Bacteriorhodopsin (bR) is a light‐driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio‐opsin (bO), the 248‐amino acid apo protein, and all‐trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl‐terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP‐sbO‐1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of ∼75% pure MBP‐sbO‐lD4 was obtained from 1 L of culture. MBP‐sbO‐lD4 formed high molecular weight (>2,000 kDa) oligomers that were water‐soluble. The synthetic bO with the 1D4 tag (sbO‐1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO‐1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP‐sbO‐1D4 and sbO‐1D4 were regenerated with all‐trans retinal and purified to homogeneity. In general, 6–10 mg of sbR‐1D4 and 52 mg of MBP‐sbR‐1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR‐lD4, MBP‐sbR‐lD4, and bR from H. salinarium.
Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli β‐galactosidase
Authors: Ayling Amanda , Baneyx François
Published: July 2010

We have studied the effect of the components of the GroE molecular chaperone machine on the refolding of the Escherichia coli enzyme β‐galactosidase, a tetrameric protein whose 116‐kDa protomers should not completely fit within the central cavity of the GroEL toroid. In the absence of other additives, GroEL formed a weak complex with chemically denatured β‐galactosidase, reduced its propensity to aggregate, and increased the recovery yields of active enzyme twofold without altering its folding pathway. When present together with the chaperonin, ATP —and to a lesser extent AMP‐PNP —reduced the recovery yields and led to the resumption of aggregation. The use of the complete chaperonin system (GroEL, GroES, and ATP) eliminated the GroEL‐mediated increase in recovery and folding proceeded less efficiently than in buffer alone. This unusual behavior can be explained in terms of a chaperonin “buffering” effect and the different affinities of GroE complexes for denatured β‐galactosidase.
A large compressibility change of protein induced by a single amino acid substitution
Authors: Gekko Kunihiko , Tamura Youjiro , Ohmae Eiji , Hayashi Hideyuki , Kagamiyama Hiroyuki , Ueno Hiroshi
Published: July 2010

The adiabatic compressibility (βs) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the βs values of both DHFR and AspAT were influenced markedly by the mutations at glycine‐121 and valine‐39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.
Complement factor D, a novel serine protease
Authors: Volanakis John E. , Narayana Sthanam V.L.
Published: July 2010

Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.
Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin
Authors: Marchot Pascale , Ravelli Raimond B.G. , Raves Mia L. , Bourne Yves , Vellom Daniel C. , Kanter Joan , Camp Shelley , Sussman Joel L. , Taylor Palmer
Published: July 2010

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl‐terminal end, was generated from a cDNA encoding the glycophospholipid‐linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one‐step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin‐mAChE complex. Complexes of two distinct fasciculins, Fasl‐mAChE and Fas2‐mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm × 0.5 mm × 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X‐ray data collected on Fas2‐mAChE crystals identified the space group as P6122 or P6522 with unit cell dimensionsa = b= 75.5 Å,c= 556 Å, giving aVmvalue of 3.1 Å3/Da (or 60% of solvent), consistent with a single molecule of Fas2‐AChE complex (72 kDa) per asymmetric unit. The complex Fasl‐mAChE crystallizes in the same space group with identical cell dimensions.
The domain organization of streptokinase: Nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments
Authors: Parrado Juan , Conejero‐Lara Francisco , Smith Richard A.G. , Marshall Julian M. , Ponting Christopher P. , Dobson Christopher M.
Published: July 2010

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N‐terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C‐terminal “tail.” Cleavage within the N‐terminal domain generates an N‐terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Pig‐dependent activator activity was shown to be SK27 (mass 27, 000, residues 147–380), which contains both central and C‐terminal domains, although this activity was reduced approximately 6, 000‐fold relative to that of full‐length SK. The activity of a 36, 000 mass fragment, SK36 (residues 64–380), which differs from SK27 in possessing a portion of the N‐terminal domain, was reduced to 0.1–1.0% of that of SK. Other fragments (masses 7, 000, 11, 000, 16, 000, 17, 000, 25, 000, and 26, 000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1–63), at a 100‐fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50‐ and > 130‐fold, respectively. These findings demonstrate that all of SK's three domains are essential for native‐like SK activity. The central and C‐terminal domains mediate plasminogen‐binding and active site‐generating functions, whereas the N‐terminal domain mediates an activity‐potentiating function.
The mitochondrial protein import motor: Dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis
Authors: Horst Martin , Oppliger Wolfgang , Feifel Bastian , Schatz Gottfried , Glick Benjamin S.
Published: July 2010

During protein import into mitochondria, matrix‐localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44‐mhsp70 complex functions as an ATP‐dependent “translocation motor” that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70‐driven translocation, we tested the effect of the purified ATP analogues AMP‐PNP and ATPγS on the Tim44‐mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44‐mhsp70 complex, but did block the ATP‐induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.
Developmentally expressed myosin heavy‐chain kinase possesses a diacylglycerol kinase domain
Authors: Thanos Christopher D. , Bowie James U.
Published: July 2010

In Dictyostelium, an ordered actin and myosin assembly‐disassembly process is necessary for proper development, differentiation, and motility (Yumura S, Fukui F, 1985, Nature 314(6007): 194–196; Ravid S, Spudich JA, 1989, J Biol Chem 264(25): 15144–15150), and phosphorylation of myosin heavy chains has been implicated in the myosin assembly‐disassembly process (Egelhoff TT, Lee RJ, Spudich JA, 1993, Cell 75(2):363–371). The developmentally expressed 84‐kDa myosin heavy‐chain kinase (MHCK) from Dictyostelium (Ravid S, Spudich JA, 1992, Proc Natl Acad Sci USA 89(13):5877–5881) is known to be a member of the protein kinase C (PKC) family. We have observed a rather striking homology between the large central domain of MHCK and the catalytic domain of diacylglycerol kinase (DGK), indicating that MHCK is in fact a gene fusion between a DGK and a PKC, possessing two separate kinase domains. The combined diacylglycerol kinase/myosin heavy‐chain kinase (DGK/MHCK) may therefore have dual functionality, possessing the ability to phosphorylate both protein and lipid. We present a hypothesis that DGK/MHCK can antagonize both actin and myosin assembly, as well as other cellular processes, by coordinated down regulation of signaling via myosin heavy‐chain kinase activity and diacylglycerol kinase activity.
Sequence replacements in the central β‐turn of plastocyanin
Authors: Ybe Joel A. , Hecht Michael H.
Published: July 2010

The role of β‐turns in dictating the structure of a β‐barrel protein is assessed by probing the tolerance of the central β‐turn of poplar plastocyanin to substitution by arbitrary sequences. Native plastocyanin binds copper and is colored bright blue. However, when the wild‐type Pro47‐Ser48‐Gly49‐Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure. Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type II β‐turn disrupt the native structure severely. Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild‐type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments. These mutant proteins do not bind copper. Furthermore, Cys84 forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed. These results indicate that the central β‐turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the β‐barrel structure. These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins.
2D 1H and 3D 1H‐15N NMR of zinc‐rubredoxins: Contributions of the β‐sheet to thermostability
Authors: Richie Kimberly A. , Teng Quincy , Elkin Christopher J. , Kurtz Donald M.
Published: July 2010

Based on 2D 1H‐1H and 2D and 3D 1H‐15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc‐containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N‐terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (‐), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcus furiosus (Pf Rd). The secondary structure of both wild‐type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same. However, the NMR data indicate an extension of the three‐stranded β‐sheet in the mutated Cp ZnRd to include the N‐terminal Ala residue and Glu 15, as occurs in Pf Rd. The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the β‐sheet and, in fact, throughout the mutated Rd. However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild‐type Cp Rds are much less thermostable than Pf Rd. These correlations strongly suggest that, contrary to a previous proposal [Blake PR et al., 1992, Protein Sci 1:1508–1521], the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N‐terminal strand of the β‐sheet. The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability.
1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease α‐Sarcin
Authors: Campos‐Olivas Ramon , Bruix Marta , Santoro Jorge , Rico Manuel , Pozo Alvaro Martinez Del , Lacadena Javier , Gavilanes José G.
Published: July 2010

The ribosome‐inactivating protein α‐Sarcin (αS) is a 150‐residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure‐dynamics‐function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of αS on the basis of homonuclear (1H‐1H) and heteronuclear (1H‐15N) two‐dimensional correlation spectra of a uniformly 15N‐labeled sample, and two selectively 15N‐labeled (Tyr and Phe) samples, as well as a single three‐dimensional experiment. The secondary structure of αS, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N‐terminal β‐hairpin, a short α‐helical segment, and a C‐terminal β‐sheet of five short strands arranged in a+1,+1,+ 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.
Derivation of 3D coordinate templates for searching structural databases: Application to ser‐His‐Asp catalytic triads in the serine proteinases and lipases
Authors: Wallace Andrew C. , Laskowski Roman A. , Thornton Janet M.
Published: July 2010

It is well established that sequence templates (e.g., PROSITE) and databases are powerful tools for identifying biological function and tertiary structure for an unknown protein sequence. Here we describe a method for automatically deriving 3D templates from the protein structures deposited in the Brookhaven Protein Data Bank. As an example, we describe a template derived for the Ser‐His‐Asp catalytic triad found in the serine proteases and triacylglycerol lipases. We find that the resultant template provides a highly selective tool for automatically differentiating between catalytic and noncatalytic Ser‐His‐Asp associations. When applied to nonproteolytic proteins, the template picks out two “non‐esterase” catalytic triads that may be of biological relevance. This suggests that the development of databases of 3D templates, such as those that currently exist for protein sequence templates, will help identify the functions of new protein structures as they are determined and pinpoint their functionally important regions.