bluuebunny/crossref_metadata_2025_split huggingface dataset:
π‐Turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence
Authors: Rajashankar K.R. , Ramakumar S. 
Published: July 2010

AbstractThe i + 5 → i hydrogen bonded turn conformation (7r‐turn) with the fifth residue adopting αL conformation is frequently found at the C‐terminus of helices in proteins and hence is speculated to be a “helix termination signal.” An analysis of the occurrence of i + 5 → i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C‐terminus), using coordinates of 228 protein crystal structures determined by X‐ray crystallography to better than 2.5 Å resolution is reported in this paper. Of 486 detected π‐turn conformations, 367 have the (i + 4)th residue in αL conformation, generally occurring at the C‐terminus of α‐helices, consistent with previous observations. However, a significant number (111) of π‐turn conformations occur with (i + 4)th residue in αR conformation also, generally occurring in α‐helices as distortions either at the terminii or at the middle, a novel finding. These two sets of 7r‐turn conformations are referred to by the names παL and παR‐turns, respectively, depending upon whether the (i + 4)th residue adopts αL or αR conformations. Four π‐turns, named π'αL ‐turns, were noticed to be mirror images of παL‐turns, and four more π‐turns, which have the (i + 4)th residue in β conformation and denoted as πβ‐turns, occur as a part of hairpin bend connecting twisted β‐strands. Consecutive π‐turns occur, but only with παR‐turns. The preference for amino acid residues is different in παL and παR‐turns. However, both show a preference for Pro after the C‐termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of παL‐turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in παL‐turns is mainly Gly and less often Asn. Although παR‐turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for παR‐turns, whereas good helix former Ala is not preferred. π‐Turns in peptides provide a picture of the π‐turn at atomic resolution. Only nine peptide‐based π‐turns are reported so far, and all of them belong to παL‐turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.
Chimeric hemoglobins — hybrids of human and swine hemoglobin: Assembly and stability of interspecies hybrids
Authors: Janardhan Rao M. , Seetharama Acharya A. , Manjula Belur N. , Kumar Ramesh 
Published: July 2010

AbstractTransgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely Hα2P
β
2 (H = human, P = swine). In an attempt to establish whether the absence of the swine α and human β (P
α
2H
β
2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine α and human β chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine α human β chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine α human β and the human α swine β interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P
α chain of P
α
2H
β
2 hybrid occurs in the presence of H
α chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P
α
2H
β
2 hybrid is lower compared to that of HbA. In contrast, P
β chain of H
α
2P
β
2 hybrid is refractory to exchange with H
β chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H
α
2P
β
2 is higher compared to that of HbA (H
α
2H
β
2). The swine α human β chimeric Hb undergoes subunit exchange reaction with human α‐chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra‐ and interdimeric interactions of the swine α and human β chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P
αH
β heterodimer (compared to the homodimers H
α
H
β and P
α
P
β)facilitates its segregation into the homodimers by subunit exchange reaction involving either H
α or P
β. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P
α
2
H
β
2 hybrid in transgenic swine expressing HbA.
An unusual route to thermostability disclosed by the comparison ofthermus thermophilusandescherichia coliinorganic pyrophosphatases
Authors: Salminen Tuna , Teplyakov Alexei , Kankare Jussi , Cooperman Barry S. , Lahti Reijo , Goldman Adrian 
Published: July 2010

AbstractThe structures ofEscherichia colisoluble inorganic pyrophosphatase (E‐PPase) andThermus thermophilussoluble inorganic pyrophosphatase (T‐PPase) have been compared to find the basis for the superior thermostability of T‐PPase. Both enzymes are D3hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T‐PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C‐terminal part of the protein, and more long‐range ion pairs from the C‐terminal helix to the rest of the molecule. Apart from the first five residues, the three‐dimensional structures of E‐PPase and T‐PPase monomers are very similar.The one striking difference, however, is in the oligomeric interactions. In comparison with an E‐PPase monomer, each T‐PPase monomer is skewed by about 1 Å in thexyplane, is 0.3 Å closer to the center of the hexamer in thezdirection, and is rotated by approximately 7° about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid‐body motion has the effect that the hexamer is more tightly packed in T‐PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T‐PPase over E‐PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid‐body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.
Improving the quality of NMR and crystallographic protein structures by means of a conformational database potential derived from structure databases
Authors: Kuszewski John , Gronenborn Angela M. , Clore G. Marius 
Published: July 2010

AbstractA new conformational database potential involving dihedral angle relationships in databases of high‐resolution highly refined protein crystal structures is presented as a method for improving the quality of structures generated from NMR data. The rationale for this procedure is based on the observation that uncertainties in the description of the nonbonded contacts present a key limiting factor in the attainable accuracy of protein NMR structures and that the nonbonded interaction terms presently used have poor discriminatory power between highland low‐probability local conformations. The idea behind the conformational database potential is to restrict sampling during simulated annealing refinement to conformations that are likely to be energetically possible by effectively limiting the choices of dihedral angles to those that are known to be physically realizable. In this manner, the variability in the structures produced by this method is primarily a function of the experimental restraints, rather than an artifact of a poor nonbonded interaction model. We tested this approach with the experimental NMR data (comprising an average of about 30 restraints per residue and consisting of interproton distances, torsion angles, 3JHNα coupling constants, and 13C chemical shifts) used previously to calculate the solution structure of reduced human thioredoxin (Qin J, Clore GM, Gronenborn AM, 1994, Structure 2:503–522). Incorporation of the conformational database potential into the target function used for refinement (which also includes terms for the experimental restraints, covalent geometry, and nonbonded interactions in the form of either a repulsive, repulsive‐attractive, or 6–12 Lennard‐Jones potential) results in a significant improvement in various quantitative measures of quality (Ramachandran plot, side‐chain torsion angles, overall packing). This is achieved without compromising the agreement with the experimental restraints and the deviations from idealized covalent geometry that remain within experimental error, and the agreement between calculated and observed 1H chemical shifts that provides an independent NMR parameter of accuracy. The method is equally applicable to crystallographic refinement, and should be particular useful during the early stages of either an NMR or crystallographic structure determination and in cases where relatively few experimental restraints can be derived from the measured data (due, for example, to broad lines in the NMR spectra or to poorly diffracting crystals).
The proteins encoded by the rbs operon of escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA
Authors: Barroga Charlene F. , Zhang Huide , Wajih Nadeem , Bouyer James H. , Hermodson Mark A. 
Published: July 2010

AbstractThe nucleotide‐binding component of the high‐affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7–7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5′‐FSBA and 8‐azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low‐level ATPase activity with a Km of about 140 μM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.
Substrate‐induced conformational changes in yeast 3‐phosphoglycerate kinase monitored by fluorescence of single tryptophan probes
Authors: Cheung Chia‐Wei , Mas Maria T. 
Published: July 2010

Abstract3‐Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of 3‐phosphoglycerate (3‐PG) and ATP to 1,3‐diphosphoglycerate (1,3‐diPG) and ADP in the presence of magnesium ions. PGK is a single polypeptide chain arranged in two domains, with an active site located in the interdomain cleft. The large distance between the binding sites for 3‐PG and ATP, deduced from the crystallographic structures of the binary complexes, gave rise to the hypothesis that this enzyme undergoes a hinge‐bending domain motion from open to closed conformation during catalysis. However, no direct experimental evidence exists for the “closed” conformation in the presence of both substrates. In this study, several PGK mutants with single tryptophans placed in various locations were used as intrinsic fluorescent probes to examine the extent and delocalization of conformational changes induced by the binding of 3‐PG, 1,3‐diPG, ADP, ATP, and PNP‐AMP (nonhydrolyzable analogue of ATP), and by 3‐PG and PNP‐AMP together. The results showed that only the probes situated in the hinge and in parts of each domain close to the hinge reflect substrate‐induced conformational changes. Binding of substrates to one domain was found to induce spectral perturbation of the probes in the opposite domain, indicating a transmission of conformational changes between the domains. A combination of both substrates generated much larger fluorescence changes than the individual substrates. The binding constants were determined for each substrate using probes situated in different locations.
Solution structure of a unique C5a semi‐synthetic antagonist: Implications in receptor binding
Authors: Zhang Xiaolu , Boyar William , Gonnella Nina C. , Galakatos Nicholas 
Published: July 2010

AbstractThe tertiary structure of a unique C5a receptor antagonist was determined by two‐dimensional NMR spectroscopy. The core domain of this 8‐kDa antagonist exists as an antiparallel helical bundle, similar to recombinant human (rh)‐C5a. However, unlike C5a, the antagonist's C terminus was found to be conformationally restricted along a groove between helices one and four in the core domain. This conformational restriction situates C‐terminal D‐Arg 75 in a wedge between core residues Arg 46 and His 15. Correlation of the antagonist's tertiary structure with point mutation analysis revealed the formation of a positively charged contiguous contact surface comprised of D‐Arg 75, Arg 46, Lys 49, and His 15. The significance of this surface in generating antagonist properties implies a single binding site with the C5a receptor and provides a structural template for drug design.
Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases
Authors: Marco Stefania Di , Fendrich Gabriele , Knecht Rene , Strauss Andre , Pohlig Gabriele , Heim Jutta , Priestle John P. , Grütter Markus G. , Sommerhoff Christian P. 
Published: July 2010

AbstractA synthetic gene coding for the 55‐amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast α‐factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N‐terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N‐terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 Å and 2.4 Å, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.
Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala‐activated protein C mediated by factor Va
Authors: Gale Andrew J. , Sun Xi , Heeb Mary J. , Griffin John H. 
Published: July 2010

AbstractThe human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and Villa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the “activated” S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma‐derived and wild‐type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R α1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half‐life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506‐FVa compared with normal Arg 506‐FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.
Analysis of the structure and stability of omega loop A replacements in yeast iso‐1‐cytochrome c
Authors: Fetrow Jacquelyn S. , Horner Scott R. , Oehrl Wolf , Schaak Diane L. , Boose Terry L. , Burton Randall E. 
Published: July 2010

AbstractOmega (Ω)‐loop A, residues 18–32 in wild‐type yeast iso‐1‐cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low‐temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native‐like cytochrome c conformation, but are less stable than the wild‐type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single‐site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.
Introduction of a [4Fe‐4S (S‐cys)4]+1,+2iron‐sulfur center into a four‐α helix protein using design parameters from the domain of the Fxcluster in the Photosystem I reaction center
Authors: Scott M. Paul , Biggins John 
Published: July 2010

AbstractWe describe the insertion of an iron‐sulfur center into a designed four α‐helix model protein. The model protein was re‐engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main‐chain directly analogous to the domain predicted to ligand the interpeptide [4Fe‐4S (S‐cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four α‐helix model. The holoprotein was characterized spectroscopically after insertion of the iron‐sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe‐4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron‐sulfur center in the model holoprotein was determined via redox titration and shown to be −422 mV (pH 8.3,n= 1). The results support proposals advanced for the structure of the domain of the [4Fe‐4S] Fxcluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fxcluster is most likely due to factors in the protein environment of Fxrather than the identity of the residues proximal to the coordinating ligands.
Intestinal fatty acid binding protein: A specific residue in one turn appears to stabilize the native structure and be responsible for slow refolding
Authors: Kim Keehyuk , Frieden Carl , Ramanathan Ragulan 
Published: July 2010

AbstractThe intestinal fatty acid binding protein is one of a class of proteins that are primarily β‐sheet and contain a large interior cavity into which ligands bind. A highly conserved region of the protein exists between two adjacent antiparallel strands (denoted as D and E in the structure) that are not within hydrogen bonding distance. A series of single, double, and triple mutations have been constructed in the turn between these two strands. In the wild‐type protein, this region has the sequence Leu 64/Gly 65/Val 66. Replacing Leu 64 with either Ala or Gly decreases the stability and the midpoint of the denaturation curve somewhat, whereas mutations at Gly 65 affect the stability slightly, but the protein folds at a rate similar to wild‐type and binds oleate. Val 66 appears not to play an important role in maintaining stability. All double or triple mutations that include mutation of Leu 64 result in a large and almost identical loss of stability from the wild‐type. As an example of the triple mutants, we investigated the properties of the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. As measured by the change in intrinsic fluorescence, this mutant (and similar triple mutants lacking leucine at position 64) folds much more rapidly than wild‐type. The mutant, and others that lack Leu 64, have far‐UV CD spectra similar to wild‐type, but a different near‐UV CD spectrum. The folded form of the protein binds oleate, although less tightly than wild‐type. Hydrogen/deuterium exchange studies using electrospray mass spectrometry indicate many more rapidly exchangeable amide protons in the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. We propose that there is a loss of defined structure in the region of the protein near the turn defined by the D and E strands and that the interaction of Leu 64 with other hydrophobic residues located nearby may be responsible for (1) the slow step in the refolding process and (2) the final stabilization of the structure. We suggest the possibility that this region of the protein may be involved in both an early and late step in refolding.