bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis and characterization of histidine‐phosphorylated peptides
Authors: Medzihradszky Katalin F. , Phillipps Nancy J. , Senderowicz Lionel , Wang Ping , Turck Christoph W. 
Published: February 2009

AbstractPosttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine‐containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.
The crystal structure of human α‐thrombin complexed with LY178550, a nonpeptidyl, active site‐directed inhibitor
Authors: Chirgadze Nickolay Y. , Sall Daniel J. , Klimkowski Valentine J. , Clawson David K. , Briggs Steve L. , Hermann Robert , Smith Gerald F. , Gifford‐Moore Donetta S. , Wery Jean‐Pierre 
Published: February 2009

AbstractThe crystal structure of human α‐thrombin in complex with LY178550, a nonpeptidyl, active site‐directed inhibitor, has been solved to 2.07 α resolution by the method of X‐ray crystallography. The final model of the complex has a crystallographic R‐value of 21.5% (Rfree = 23.1%) with 0.014 Å and 2.4° standard deviation from ideal bond lengths and angles, respectively. Well‐defined electron density was observed for the inhibitor in the active site. The inhibitor binds to the active site in an L‐shaped manner, mimicking the bound conformation of the tripeptide arginal series of thrombin inhibitors (Chirgadze NY et al., 1992, American Crystallographic Association Meeting 20:116 [Abstr. PB311]). The basic amidine of LY178550 forms a salt bridge with Asp 189 within the specificity pocket, while the 4‐benzylpiperidine side chain engages in a number of hydrophobic interactions at the S2 and S3 binding sites. The inhibitor does not interact in any fashion with the active site sequence Ser 214‐Gly 216, as occurs with many of the inhibitors studied previously. The indole N‐H of the inhibitor forms a hydrogen bond to the γ‐oxygen of the catalytic serine (Ser 195).
The crystal structure of the mouse glandular kallikrein‐13 (prorenin converting enzyme)
Authors: Timm David E. 
Published: February 2009

AbstractA crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK‐13) has been determined at 2.6‐Å resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin‐converting enzyme and cleaves submandibular gland Ren‐2 prorenin to yield active renin. The mGK‐13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and an active site located in the cleft between two β‐barrel domains. The mGK‐13 structure reveals for the first time an ordered kallikrein loop conformation containing a short 310 helix. This loop is disordered in the related porcine pancreatic kallikrein and rat submandibular tonin structures. The kallikrein loop is in close spatial proximity to the active site and is also involved in a dimeric arrangement of mGK‐13. The catalytic specificity of mCK‐13 for Ren‐2 prorenin was studied by modeling a prorenin‐derived peptide into the active site of mGK‐13. This model emphasizes two electronegative substrate specificity pockets on the mGK‐13 surface, which could accommodate the dibasic P2 and P1 residues at the site of prorenin cleavage by mGK‐13.
Structure‐based subsite specificity mapping of human cathepsin D using statine‐based inhibitors
Authors: Majer Pavel , Collins Jack R. , Gulnik Sergei V. , Erickson John W. 
Published: February 2009

AbstractHuman cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D‐pepstatin A complex, a series of statine‐containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P'2) with the aim of mapping the cathepsin D subsite preferences. The experimentally obtained SAR data were correlated on the basis of molecular modeling. Side‐chain preferences for the peptidomimetic inhibitors differed from those found previously using peptide substrates (Scarborough PE et al., 1993, Protein Sci 2:264‐276). In addition, the effects of single side‐chain modifications were often nonadditive. Structure‐activity relationships, modeling, and thermodynamic analysis indicated that entropy plays a major stabilizing role in inhibitor binding to cathepsin D.
Structural studies of receptor binding by cholera toxin mutants
Authors: Merritt Ethan A. , Sarfaty Steve , Hol WIM G.J. , Jobling Michael G. , Holmes Randall K. , Chang T. , Hirst Timothy R. 
Published: February 2009

AbstractThe wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B‐pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain.We now report the structure determination and 2.3‐Å refinement of the CTB mutant Gly 33 → Arg complexed with the Gm1 oligosaccharide, as well as the 2.2‐Å refinement of a Gly 33 → Asp mutant of the closely related Escherichia coli heat‐labile enterotoxin B‐pentamer (LTB). Two of the five receptor binding sites in the Gly 33 → Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 Å) determination and refinement of the wild‐type CTB:GM1oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild‐type and Gly 33 → Arg mutant complexes.The protein conformation observed for the binding‐deficient Gly 33 → Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33 → Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self‐affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.
Epitope and mimotope for an antibody to the Na, K‐ATPase
Authors: Pacholczyk Tadeusz , Sweadner Kathleen J. 
Published: February 2009

AbstractThe epitope of a monoclonal antibody specific for the α2 isoform of the Na,K‐ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N‐chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K‐ATPase N‐terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K‐ATPase. Full‐length cDNA for the Na,K‐ATPase was randomly fragmented and cloned into β‐galactosidase to create a λgt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N‐terminus. Chimeras of Na,K‐ATPase α1 and α2 narrowed down the epitope to 14‐19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K‐ATPase α2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or β‐galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N‐terminus, where the Na,K‐ATPase isofonns differ most.
Mutational analysis of the BPTI folding pathway: II. Effects of aromatic → leucine substitutions on folding kinetics and thermodynamics
Authors: Zhang Jian‐Xin , Goldenberg David P. 
Published: February 2009

AbstractThe rates of the individual steps in the disulfide‐coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide‐bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically‐preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild‐type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.
Cloning, purification, and preliminary characterization by circular dichroism and NMR of a carboxyl‐terminal domain of the bacteriophage P22 scaffolding protein
Authors: Parker Matthew H. , Prevelige Peter E. , Jablonsky Michael , Casens Sherwood , Sampson Laura , Krishna N. Rama 
Published: February 2009

AbstractAssembly of double‐stranded DNA viruses and bacteriophages involves the polymerization of several hundred molecules of coat protein, directed by an internal scaffolding protein. A 163‐amino acid carboxyl‐terminal fragment of the 303‐amino acid bacteriophage P22 scaffolding protein was cloned, over‐expressed, and purified. This fragment is active in procapsid assembly reactions in vitro. The circular dichroism spectrum of the fragment, as well as the 1D‐NMR and 15N‐1H HSQC spectra of the uniformly‐labeled protein, indicate that stable secondary structure elements are present. Determination of the three dimensional packing of these elements into the folded scaffolding protein fragment is underway. Structure‐based drug design targeted at structural proteins required for viral assembly may have potential as a therapeutic strategy.
Three‐dimensional structures and contexts associated with recurrent amino acid sequence patterns
Authors: Han Karen E , Bystroff Christopher , Baker David 
Published: February 2009

AbstractWe have used cluster analysis to identify recurring sequence patterns that transcend protein family boundaries. A subset of these patterns occur predominantly in a single type of local structure in proteins. Here we characterize the three‐dimensional structures and contexts in which these sequence patterns occur, with particular attention to the interactions responsible for their structural selectivity.
Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: Sequence analysis and mutagenesis identify receptor binding epitopes
Authors: Jerva L. Fred , Lolis Elias , Sullivan Gail 
Published: February 2009

AbstractMurine macrophage inflammatory protein‐2 (MIP‐2), a member of the α‐ chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the α‐chemokines, such as interleukin‐8, gro‐α/MGSA, and neutrophil activating peptide‐2 (NAP‐2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP‐2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP‐2 exhibits a highly ordered secondary structure consistent with the α/β structures of other chemokines. Recombinant MIP‐2 is chemotactic for human and murine neutrophils and up‐regulates cell surface expression of Mac‐1. MIP‐2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP‐2 to the human types A and B IL‐8 receptors and the murine homologue of the IL‐8 receptor. MIP‐2 displays low‐affinity binding to the type A IL‐8 receptor (Kd >120 nM) and high‐affinity binding to the type B IL‐8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three‐dimensional structure of IL‐8 and sequence analysis of six chemokines (IL‐8, gro‐α, NAP‐2, ENA‐78, KC, and MIP‐2) that display highaffinity binding to the IL‐8 type B receptor are used to identify an extended N‐terminal surface that interacts with this receptor. Two mutants of MIP‐2 establish that this region is also involved in binding and activating the murine homologue of the IL‐8 receptor. Differences in the sequence between IL‐8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL‐8 for binding to the type A receptor.
Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A
Authors: Catanzano Francesca , Barone Guido , Graziano Giuseppe , Capasso Sante 
Published: February 2009

AbstractSelective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature‐induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0‐6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 °C of denaturation temperature and 65 kJ mol −1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X‐ray three‐dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine‐2′‐monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.
Coupling backbone flexibility and amino acid sequence selection in protein design
Authors: Su Alyce , Mayo Stephen L. 
Published: February 2009

AbstractUsing a protein design algorithm that considers side‐chain packing quantitatively, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G β1 domain (Gβ1). Concerted backbone motion was introduced by varying Gβ1′s supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally and showed that core variants containing as many as 6 of 10 possible mutations retain native‐like properties. This result demonstrates that backbone flexibility can be combined explicitly with amino acid side‐chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of Gβ1's native supersecondary structure parameter values.
The membrane topology of the amino‐terminal domain of the red cell calcium pump
Authors: Castello Pablo R. , Flecha F. Luis González , Caride Ariel J. , ández Horacio N.Fern , Delfino José M. , Rossi JUAN P.F.C. 
Published: February 2009

AbstractA systematic study of the membrane‐associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3‐(trifluorometnyl)‐3‐(m‐[ 125I]iodophenyl)‐diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane‐bound probe, [3H]bis‐phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light‐dependent labeling of an Mr 135,000‐140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS‐PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane‐embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N‐terminal domain of the Ca2+ pump. A labeled peptide of Mr 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino‐terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1‐322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with α‐helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N‐terminus of the Ca2+ pump.
The effect of denaturants on protein structure
Authors: Dunbar Jennifer , Yennawar Hemant P. , Banerjee Soojay , Luo Jiabin , Farber Gregory K. 
Published: February 2009

AbstractVirtually all studies of the protein‐folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X‐ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. J Mol Biol 226:491‐505).
Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization domain from cartilage matrix protein in oxidized and reduced forms
Authors: Wiltscheck Ronald , Dames Sonja A. , Alexandrescu Andrei T. , Kammerer Richard A. , Schulthess Therese , Blommers MARCEL J.J. , Engel JŮRgen 
Published: February 2009

AbstractThe C‐terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43‐residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence‐specific assignments for 1H‐, 15N‐, and 13C‐NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY‐HSQC, and HCCH‐TOCSY spectra. A stretch of α‐helix encompassing five heptad repeats (35 residues) has been identified from intra‐chain HN‐HN and HN‐Hα NOE connectivities, 3 JHNHα coupling constants, and chemical shift indices. The α‐helix begins immediately downstream of inter‐chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C‐terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter‐chain disulfide bonds that flank the N‐terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N‐terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed.
Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide‐binding protein
Authors: Smith Virginia F. , Randall Linda L. , Hardy SIMON J. S. 
Published: February 2009

AbstractChaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide‐binding protein. Similar studies performed on the maltose‐binding and galactose‐binding proteins revealed centrally positioned binding frames of ∼160 aminoacyl residues. The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein. We propose modes of binding to account for the data.