bluuebunny/crossref_metadata_2025_split huggingface dataset:
The structural alignment between two proteins: Is there a unique answer?
Authors: Godzik Adam 
Published: February 2009

AbstractStructurally similar but sequentially unrelated proteins have been discovered and rediscovered by many researchers, using a variety of structure comparison tools. For several pairs of such proteins, existing structural alignments obtained from the literature, as well as alignments prepared using several different similarity criteria, are compared with each other. It is shown that, in general, they differ from each other, with differences increasing with diminishing sequence similarity. Differences are particularly strong between alignments optimizing global similarity measures, such as RMS deviation between Cα atoms, and alignments focusing on more local features, such as packing or interaction pattern similarity. Simply speaking, by putting emphasis on different aspects of structure, different structural alignments show the unquestionable similarity in a different way. With differences between various alignments extending to a point where they can differ at all positions, analysis of structural similarities leads to contradictory results reported by groups using different alignment techniques.The problem of uniqueness and stability of structural alignments is further studied with the help of visualization of the suboptimal alignments. It is shown that alignments are often degenerate and whole families of alignments can be generated with almost the same score as the “optimal alignment.” However, for some similarity criteria, specially those based on side‐chain positions, rather than Cα positions, alignments in some areas of the protein are unique. This opens the question of how and if the structural alignments can be used as “standards of truth” for protein comparison.
Ala‐insertion scanning mutagenesis of the glycophorin a transmembrane helix: A rapid way to map helix‐helix interactions in integral membrane proteins
Authors: Mingarro Ismael , Whitley Paul , Heijne Gunnar Von , Lemmon Mark A. 
Published: February 2009

AbstractAlanine insertions into the glycophorin A transmembrane helix are found to disrupt helix‐helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala‐insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.
Novel isolation method and structural stability of a eukaryotic chaperonin: The TCP‐1 ring complex from rabbit reticulocytes
Authors: Norcum Mona Trempe 
Published: February 2009

AbstractIn the course of removing a contaminant from preparations of aminoacyl‐tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin‐agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino‐terminal sequencing, and ATP‐dependent refolding of rhodanese and actin.Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the “characteristic” ring and four‐stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate‐containing views are observed. The particle structure is strikingly resistant to physical forces (long‐term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KC1 or LiCl), and pH changes (9‐6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KC1 or deoxycholate reduces TRiC folding activity.
Conformational stability of apoflavodoxin
Authors: Genzor Carlos G. , Gómez‐Moreno Carlos , Sancho Javier , Beldarraín Alejandro , López‐Lacomba José Luis , Cortijo Manuel 
Published: February 2009

AbstractFlavodoxins areα/βproteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin fromAnabœnaPCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near‐ultraviolet (UV) circular dichroism (CD) and high‐field1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two‐state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5‐2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far‐UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two‐state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding ofα/βproteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.
Membrane‐bound states of α‐lactalbumin: Implications for the protein stability and conformation
Authors: Cawthern Kevin M. , Permyakov Eugene , Berliner Lawrence J. 
Published: February 2009

Abstractα‐Lactalbumin (α‐LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC α‐LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC‐α‐LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein‐DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154‐166). On the other hand, above the protein transition (70 °C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC‐α‐LA complex showed a distinct fluorescence thermal transition between 40 and 60 °C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154‐166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan‐containing surface is covered by the lipid phase of the bilayer.
Experimentally observed conformation‐dependent geometry and hidden strain in proteins
Authors: Karplus P. Andrew 
Published: February 2009

AbstractA database has been compiled documenting the peptide conformations and geometries from 70 diverse proteins refined at 1.75 Å or better. Analysis of the well‐ordered residues within the database shows ϕ, Ψ‐distributions that have more fine structure than is generally observed. Also, clear evidence is presented that the peptide covalent geometry depends on conformation, with the interpeptide N‐Cα‐C bond angle varying by nearly ±5 degrees from its standard value. The observed deviations from standard peptide geometry are greatest near the edges of well‐populated regions, consistent with strain occurring in these conformations. Minimization of such hidden strain could be an important factor in thermostability of proteins. These empirical data describing how equilibrium peptide geometry varies as a function of conformation confirm and extend quantum mechanics calculations, and have predictive value that will aid both theoretical and experimental analyses of protein structure.
Crystallization, X‐ray studies, and site‐directed cysteine mutagenesis of the DNA‐binding domain of OmpR
Authors: Martinez‐Hackert Erik , Berman Helen M. , Harlocker Susan , Inouye Masayori , Stock Ann M. 
Published: February 2009

AbstractA C‐terminal fragment of the transcription factor OmpR has been crystallized using the sitting drop vapor‐diffusion method. Crystals belong to the trigonal spacegroup P3n12 with cell dimensions a = b = 54.4 Å, c = 135.5 Å, and γ = 120.00°. A second crystal form has been obtained by soaking this crystal form in a cryo‐buffer and flash‐cooling to 108 K in a cold nitrogen stream. Crystals belong to the trigonal space‐group P3n12 with cell dimensions a = b = 108.07 Å, c = 131.81Å, and γ = 120.00°. Both crystal forms diffract to at least 2.3 Å at a synchrotron light source. Single‐site cysteine mutations have been introduced to provide mercury‐binding sites for multiple isomorphous replacement.
Protein folding in the endoplasmic reticulum: Lessons from the human chorionic gonadotropin β subunit
Authors: Ruddon Raymond W. , Sherman Simon A. , Bedows Elliott 
Published: February 2009

AbstractThere have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin β subunit (hCG‐β), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG‐β fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the β subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects.
Structure of equine infectious anemia virus proteinase complexed with an inhibitor
Authors: Gustchina Alla , Kervinen Jukka , Powell David J. , Zdanov Alexander , Kay John , Wlodawer Alexander 
Published: February 2009

AbstractEquine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus‐encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X‐ray structure of a complex of the I54G mutant of EIAV PR with the inhibitor HBY‐793 was solved at 1.8 Å resolution and refined to a crystallographic R‐factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second α‐helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate‐binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme‐ligand interactions at the periphery of the active site as well.
A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1
Authors: Doumen Jan , Gonciarz Malgorzata , Zegers Ingrid , Loris Remy , Wyns Lode , Steyaert Jan 
Published: February 2009

AbstractThe function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site‐directed mutagenesis and X‐ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75‐fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild‐type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2′‐GMP are restricted to the active site. Repositioning of 2′‐GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.
RhNGF slow unfolding is not due to proline isomerization: Possibility of a cystine knot loop‐threading mechanism
Authors: de Young Linda R. , Burton Louis E. , Liu Jun , Powell Michael F. , Schmelzer Charles H. , Skelton Nicholas J. 
Published: February 2009

AbstractThe unfolding of recombinant human β‐NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 °C. Fluorescence spectroscopy, size‐exclusion and reversed‐phase HPLC, ultra‐centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little β‐sheet structure, but retained residual structure in the tryptophan indole and high‐field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half‐life of 23 h at 25 °C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N‐terminus. A model for the slow unfolding reaction is proposed where the N‐terminus threads through the cystine knot to form M2, a loop‐threading reaction, increasing the conformational freedom of the denatured state.
Solid‐state NMR studies of the prion protein H1 fragment
Authors: Heller Jonathan , Kolbert Andrew C. , Larsen Russell , Ernst Matthias , Bekker Tatiana , Baldwin Michael , Prusiner Stanley B. , Pines Alexander , Wemmer David E. 
Published: February 2009

AbstractConformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation‐dependent chemical‐shift measurements and rotational‐resonance distance measurements to analyze the conformation of solid‐state peptides lacking long‐range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid‐state NMR spectra of specifically 13C‐enriched samples of H1, residues 109–122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross‐polarization and magic‐angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a β‐sheet conformation in the region corresponding to residues 112–121, whereas samples lyophilized from hexa‐fluoroisopropanol display shifts indicative of α‐helical secondary structure in the region corresponding to residues 113–117. Complete conversion to the helical conformation was not observed and conversion from α‐helix back to β‐sheet, as inferred from the solid‐state NMR spectra, occurred when samples were exposed to water. Rotational‐resonance experiments were performed on seven doubly 13C‐labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly β‐strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.
Models for the 310‐helix/coil, π‐helix/coil, and α‐helix/310‐helix/coil transitions in isolated peptides
Authors: Rohl Carol A. , Doig Andrew J. 
Published: February 2009

AbstractModels for the 310‐helix/coil and π‐helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N‐cap position, a C‐cap position, or the reference coil conformation. The models for α‐helix formation and 310‐helix formation have also been combined to describe a three‐state equilibrium in which α‐helical, 310‐helical, and coil conformations are populated. The results are compared with the modified Lifson‐Roig theory for the α‐helix/coil equilibrium. The comparison accounts for the experimental observations that 310‐helices tend to be short and π‐helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 310‐helices and mixed 310‐/α‐helices.
Topology prediction for helical transmembrane proteins at 86% accuracy–Topology prediction at 86% accuracy
Authors: Rost Burkhard , Fariselli Piero , Casadio Rita 
Published: February 2009

AbstractPreviously, we introduced a neural network system predicting locations of transmembrane helices (HTMs) based on evolutionary profiles (PHDhtm, Rost B, Casadio R, Fariselli P, Sander C, 1995, Protein Sci 4:521–533). Here, we describe an improvement and an extension of that system. The improvement is achieved by a dynamic programming‐like algorithm that optimizes helices compatible with the neural network output. The extension is the prediction of topology (orientation of first loop region with respect to membrane) by applying to the refined prediction the observation that positively charged residues are more abundant in extra‐cytoplasmic regions. Furthermore, we introduce a method to reduce the number of false positives, i.e., proteins falsely predicted with membrane helices. The evaluation of prediction accuracy is based on a cross‐validation and a double‐blind test set (in total 131 proteins). The final method appears to be more accurate than other methods published: (1) For almost 89% (π3%) of the test proteins, all HTMs are predicted correctly. (2) For more than 86% (π3%) of the proteins, topology is predicted correctly. (3) We define reliability indices that correlate with prediction accuracy: for one half of the proteins, segment accuracy raises to 98%; and for two‐thirds, accuracy of topology prediction is 95%. (4) The rate of proteins for which HTMs are predicted falsely is below 2% (π1%). Finally, the method is applied to 1,616 sequences of Haemophilus influenzae. We predict 19% of the genome sequences to contain one or more HTMs. This appears to be lower than what we predicted previously for the yeast VIII chromosome (about 25%).
Covalent structure of the flavoprotein subunit of the flavocytochrome c: Sulfide dehydrogenase from the purple phototrophic bacterium chromatium vinosum
Authors: Driessche Gonzalez Van , Koh Monjoo , Van Beeumen Jozef J. , Chen Zhi‐Wei , Mathews F. Scott , Meyer Terry E. , Bartsch Robert G. , Cusanovich Michael A. 
Published: February 2009

AbstractThe amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c—sulftde dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three‐dimensional structure determination (Chen Z et al., 1994, Science 266:430–432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 ± 44 Da measured by LDMS. There are six cysteine residues, among which Cys 42 provides the site of covalent attachment of the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the FAD. The flavoprotein subunit of FCSD is most closely related to glutathione reductase (GR) in three‐dimensional structure and, like that protein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in sequence is not significantly greater than for random comparisons. The first domain binds FAD in both proteins. Domain 2 of GR is the site of NADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfur compounds. Domains 1 and 2 of FCSD, as of GR, are homologous to one another and represent an ancient gene doubling. The third domain provides the dimerization interface for GR, but is the site of binding of the cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite adduct formation and decay, have now been identified from the three‐dimensional structure and the sequence as Cys 161/Cys 337 disulfide, Tip 391, Glu 167, and the positive end of a helix dipole.