bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prediction of protein complexes using empirical free energy functions
Authors: Weng Zhiping , Vajda Sandor , Delisi Charles 
Published: July 2010

AbstractA long sought goal in the physical chemistry of macromolecular structure, and one directly relevant to understanding the molecular basis of biological recognition, is predicting the geometry of bimolecular complexes from the geometries of their free monomers. Even when the monomers remain relatively unchanged by complex formation, prediction has been difficult because the free energies of alternative conformations of the complex have been difficult to evaluate quickly and accurately. This has forced the use of incomplete target functions, which typically do no better than to provide tens of possible complexes with no way of choosing between them. Here we present a general framework for empirical free energy evaluation and report calculations, based on a relatively complete and easily executable free energy function, that indicate that the structures of complexes can be predicted accurately from the structures of monomers, including close sequence homologues. The calculations also suggest that the binding free energies themselves may be predicted with reasonable accuracy. The method is compared to an alternative formulation that has also been applied recently to the same data set. Both approaches promise to open new opportunities in macromolecular design and specificity modification.
High level expression and crystallization of recombinant human cathepsin S
Authors: Brömme Dieter , Mcgrath Mary E. 
Published: July 2010

AbstractWe have expressed active human cathepsin S to 60 mg/L in Sf9 cells using a baculovirus system. Production of milligram quantities has facilitated crystallographic studies to determine the structure of this enzyme, which has unique properties among lysosomal cysteine proteinases. Recombinant, irreversibly inhibited cathepsin S was crystallized from ammonium phosphate at 17 °C. The crystals diffract to at least 2.3 Å, and belong to the orthorhombic crystal system with a primitive lattice. Approximate cell dimensions are: a = 37.7 Å, b = 73.9 Å, and c = 106.7 Å. There is most likely one molecule per asymmetric unit.
Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of minibody
Authors: Zappacosta Francesca , Pessi Antonello , Bianchi Elisabetta , Venturini Sara , Sollazzo Maurizio , Tramontano Anna , Marino Gennaro , Pucci Piero 
Published: July 2010

AbstractA strategy that combines limited proteolysis experiments and mass spectrometrie analysis of the fragments generated has been developed to probe protease‐accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64‐residue protein consisting of a β‐sheet scaffold based on the heavy‐chain variable‐domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X‐ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single‐residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X‐ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.
Interaction of subtilisins with serpins
Authors: Komiyama Tomoko , Grøn Hanne , Salvesen Guy S. , Pemberton Philip A. 
Published: July 2010

AbstractSerpins are well‐characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that α1proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and α1antichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0–45 °C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.
Enzymatic and fluorescence studies of four single‐tryptophan mutants of rat testis fructose 6‐phosphate,2‐kinase:fructose 2,6‐bisphosphatase
Authors: Watanabe Fusao , Uyeda Kosaku , Jameson David M. 
Published: July 2010

AbstractIn order to determine environments around four tryptophan residues, located in the N‐terminus, in the kinase and in the phosphatase domains of rat testis Fru 6‐P,2‐kinase:Fru 2,6‐bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site‐directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild‐type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5× that of the wild‐type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild‐type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild‐type enzyme. The polarization in the presence of Fru 6‐P affected only Trp 15, which suggested that it is located near the Fru 6‐P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6‐P,2‐kinase and Fru 2,6‐bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.
Protein folding for realists: A timeless phenomenon
Authors: Shortle David , Wang Yi , Gillespie Joel R. , Wrabl James O. 
Published: July 2010

AbstractFuture research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form in the first few milliseconds of the refolding reaction. (2) The energy functions used to predict structure from sequence will always be approximations of the true energy function. One strategy to resolve both dilemmas is to view protein folding from a different perspective, one that no longer emphasizes time and unique trajectories through conformation space. Instead, free energy replaces time as the reaction coordinate, and ensembles of equilibrium states of partially folded proteins are analyzed in place of trajectories of one protein chain through conformation space, either in vitro or in silico. Initial characterization of the folding of staphylococcal nuclease within this alternative conceptual framework has led to an equilibrium folding pathway with several surprising features. In addition to the finding of two bundles of four hydrophobic segments containing both native and non‐native interactions, a gradient in relative stability of different substructures has been identified, with the most stable interactions located toward the amino terminus and the least stable toward the carboxy terminus. Hydrophobic bundles with up‐down topology and stability gradients may be two examples of numerous tactics used by proteins to facilitate rapid folding and minimize aggregation. As NMR methods for structural analysis of partially folded proteins are refined, higher resolution descriptions of the structure and dynamics of the polypeptide chain outside the native state may provide many insights into the processes and energetics underlying the self‐assembly of folded structure.
Mobile unnatural amino acid side chains in the core of staphylococcal nuclease
Authors: Wynn Richard , Harkins Paul C. , Richards Frederic M. , Fox Robert O. 
Published: July 2010

AbstractThe structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X‐ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1‐n‐propane, 1‐n‐butane, 1‐n‐pentane, and 2‐hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side chains as large as the 1‐n‐propyl cysteine disulfide can be incorporated without perturbation of the structure. This is due, in part, to cavities present in the wild‐type protein. The longest side chains are not well defined, even though they remain buried within the protein interior. These results suggest that the enthalpy‐entropy balance that governs the rigidity of protein interiors favors tight packing only weakly. Additionally, the tight packing observed normally in protein interiors may reflect, in part, the limited numbers of rotamers available to the natural amino acids.
Crystal structure of cutinase covalently inhibited by a triglyceride analogue
Authors: Longhi Sonia , Knoops‐Mouthuy Edith , Cambillau Christian , Mannesse Maurice , Verheij Hubertus M. , De Haas Gerard H. , Egmond Maarten 
Published: July 2010

AbstractCutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently. All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors, which bear little resemblance to a natural triglyceride substrate. In this article we describe the crystal structure of cutinase covalently inhibited by (R)‐1,2‐dibutyl‐carbamoylglycero‐3‐O‐p‐nitrophenylbutyl‐phosphonate, a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway. The structure, which has been solved at 2.3 Å, reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice. The overall shape of the inhibitor is that of a fork: the two dibutyl‐carbamoyl chains point towards the surface of the protein, whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn‐1 and sn‐2 chains. The sn‐3 chain is accommodated in a rather small pocket at the bottom of the active site crevice, thus providing a structural explanation for the preference of cutinase for short acyl chain substrates.
A desolvation barrier to hydrophobic cluster formation may contribute to the rate‐limiting step in protein folding
Authors: Rank Jeffrey A. , Baker David 
Published: July 2010

AbstractTo gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water‐excluding cluster relative to both the close‐packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent‐accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.
Identification of a novel phosphatase sequence motif
Authors: Stukey Joseph , Carman George M. 
Published: July 2010

AbstractWe have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP‐(X12‐54)‐PSGH‐(X31‐54))‐SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose‐6‐phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium‐containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vana‐date, a compound structurally similar to phosphate. Structure‐function analysis of the human glucose‐6‐phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.
An engineered amino‐terminal domain of yeast phosphoglycerate kinase with native‐like structure
Authors: Sherman Mark A. , Chen Yuan , Mas Maria T. 
Published: July 2010

AbstractPrevious studies have suggested that the carboxy‐terminal peptide (residues 401–415) and interdomain helix (residues 185–199) of yeast phosphoglycerate kinase, a two‐domain enzyme, play a role in the folding and stability of the amino‐terminal domain (residues 1–184). A deletion mutant has been created in which the carboxy‐terminal peptide is attached to the amino‐terminal domain (residues 1–184) plus interdomain helix (residues 185–199) through a flexible peptide linker, thus eliminating the carboxy‐terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N‐domain is soluble, monomeric, compactly folded, native‐like in structure, and capable of binding the substrate 3‐phosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine‐induced unfolding transition was the same as that of the native two‐domain protein (Cm ∼ 0.8 M). The free energy change associated with guanidine‐induced unfolding was one‐third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N‐domain and the contribution of domain‐domain interactions to the stability of PGK. These observations suggest that the C‐terminal peptide and interdomain helix are sufficient for maintaining a native‐like fold of the N‐domain in the absence of the C‐domain.
Amino acid sequence of bovine gamma E (IVa) lens crystallin
Authors: Kilby Greg W. , Sheil Margaret M. , Shaw Denis , Harding John J. , Truscott Roger J. W. 
Published: July 2010

AbstractWhen electrospray ionization mass spectrometry (ES‐MS) was used to analyze purified bovine gamma E (γIVa)‐crystallin, it yielded a relative molecular mass (Mr) of 20,955 ± 5. This mass is significantly different from that calculated from the published sequence (Mr 20,894) (White HE et al., 1989, J Mol Biol 207:217–235). Further, ES‐MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203–208). The entire protein sequence of γE crystallin has therefore been studied via a combination of ES‐MS, ES‐MS/MS, and Edman amino acid sequencing. The corrected sequence gives an Mr of 20,955.3, which matches that obtained by ES‐MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine γ‐crystallin by R. Hay (pers. comm.).
Production, crystallization, and preliminary X‐ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1‐47) of troponin I
Authors: Saijo Yumiko , Takeda Soichi , Scherer Anna , Kobayashi Tomoyoshi , Maéda Yuichiro , Taniguchi Hisaaki , Yao Min , Wakatsuki Soichi 
Published: July 2010

AbstractTroponin is a ternary protein complex consisting of subunits TnC, TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli‐expressed rabbit skeletal muscle TnC and fragment 1‐47 of TnI, which is obtained by chemical cleavage of an E. coli‐expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P31(2)21, with unit cell dimensions a = b = 48.2 Å, c = 162 Å. The asymmetric unit presumably contains one CI47 complex. Soaking with p‐chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 Å resolution, with completeness of 91% and Rmerge = 6.4%. The crystals of PCMBS‐derivative should be suitable for X‐ray studies using the multiple‐wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.
Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus
Authors: Solow Barbara , Bischoff Kenneth M. , Zylka Mark J. , Kennelly Peter J. 
Published: July 2010

AbstractWhen soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [γ‐32P]ATP, several radiolabeled polypeptides were observed following SDS‐PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35‐kDa one, whose protein‐phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35‐ and 50‐kDa phosphoproteins were purified and their amino‐terminal sequences determined. The former polypeptide's amino‐terminal sequence closely matched a conserved portion of the α‐subunit of succinyl‐CoA synthetase, which forms an acid‐labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50‐kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro‐Gly‐Thr‐Asp/Ser‐Gly‐Val/Leu‐Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6‐phosphate to donate its phosphoryl group to the protein.
X‐ray structures of three interface mutants of γB‐crystallin from bovine eye lens
Authors: Palme Stefan , Slingsby Christine , Jaenicke Rainer 
Published: July 2010

AbstractγB‐crystallin consists of two domains each comprising two “Greek key” motifs. Both domains fold independently, and domain interactions contribute significantly to the stability of the C‐terminal domain. In a previous study (Palme S et al., 1996, Protein Sci 6:1529‐1636) it was shown that Phe56 from the N‐terminal domain, a residue involved in forming a hydrophobic core at the domain interface, effects the interaction of the two domains, and therefore, the stability of the C‐terminal domain. Ala or Asp at position 56 drastically decreased the stability of the C‐terminal domain, whereas Trp had a more moderate effect. In this article we present the X‐ray structures of these interface mutants and correlate them with the stability data. The mutations do not effect the overall structure of the molecule. No structural changes are observed in the vicinity of the replaced residue, suggesting that the local structure is too rigid to allow compensations for the amino acid replacements. In the mutants γB‐F56A and ‐F56D, a solvent‐filled groove accessible to the bulk solvent is created by the replacement of the bulky Phe side chain. In γB‐F56W, the pyrrole moiety of the indole ring replaces the phenyl side chain of the wild type. With the exception of γB‐F56W, there is a good correlation between the hydrophobicity of the amino acid at position 56 according to the octanol scale and the stability of the C‐terminal domain. In γB‐F56W, the C‐terminal domain is less stable than estimated from the hydrophobicity, presumably because the ring nitrogen (Nϵ1) has no partner to form hydrogen bonds. The data suggest that the packing of hydrophobic residues in the interface core is important for domain interactions and the stability of γB‐crystallin. Apparently, for protein stability, the same principles apply for hydrophobic cores within domains and at domain interfaces.
Locally accessible conformations of proteins: Multiple molecular dynamics simulations of crambin
Authors: Caves Leo S. D. , Evanseck Jeffrey D. , Karplus Martin 
Published: July 2010

AbstractMultiple molecular dynamics (MD) simulations of crambin with different initial atomic velocities are used to sample conformations in the vicinity of the native structure. Individual trajectories of length up to 5 ns sample only a fraction of the conformational distribution generated by ten independent 120 ps trajectories at 300 K. The backbone atom conformational space distribution is analyzed using principal components analysis (PCA). Four different major conformational regions are found. In general, a trajectory samples only one region and few transitions between the regions are observed. Consequently, the averages of structural and dynamic properties over the ten trajectories differ significantly from those obtained from individual trajectories. The nature of the conformational sampling has important consequences for the utilization of MD simulations for a wide range of problems, such as comparisons with X‐ray or NMR data. The overall average structure is significantly closer to the X‐ray structure than any of the individual trajectory average structures. The high frequency (less than 10 ps) atomic fluctuations from the ten trajectories tend to be similar, but the lower frequency (100 ps) motions are different. To improve conformational sampling in molecular dynamics simulations of proteins, as in nucleic acids, multiple trajectories with different initial conditions should be used rather than a single long trajectory.