bluuebunny/crossref_metadata_2025_split huggingface dataset:
Folding proteins with a simple energy function and extensive conformational searching
Authors: Yue Kaizhi , Dill Ken A. 
Published: July 2010

AbstractWe 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

AbstractThe 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

AbstractThymidylate 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

AbstractSeveral 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

AbstractA 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

AbstractA 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

AbstractBacteriorhodopsin (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

AbstractWe 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

AbstractThe 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

AbstractFactor 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

AbstractA 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

AbstractStreptococcus 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

AbstractDuring 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

AbstractIn 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

AbstractThe 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

AbstractBased 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

AbstractThe 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.