bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational analysis of phenylalanine β85 in the valine β6 acceptor pocket during hemoglobin S polymerization
Authors: Adachim Kazuhiko , Reddy Lattupally R. , Surrey Saul , Reddy Konda S. 
Published: February 2009

AbstractHemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at β85 were made and expressed in yeast in an effort to evaluate the role of Phe‐β85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these β385 substitutions do not significantly affect heme‐globin interactions and tetramer helix content. Hb S containing Trp‐β85 had decreased oxygen affinity, whereas those with Leu‐, Ala‐, and Thr‐β85 had increased oxygen affinity. All four supersaturated β85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe‐β88, Ala‐β88, Glu‐β88, or Glu‐β85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477–17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563–31566). Leu substitution at β85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these β85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SFβ85L, Hb SFβ85A, Hb SFβ85T, and Hb sFβ85W are 0.65‐, 2.2‐, 2.5‐ and 3‐fold higher, respectively, than Hb S. These results suggest that the relative order for polymerization of β85 variants (Leu > Phe > Ala > Thr > Trp‐β85) depends on amino acid hydrophobicity rather than stereospecificity of the side chain. These findings are in contrast to previous results for β88 variants. Trp‐β85 in Hb S may affect Val‐β6 acceptor pocket size, but may still accommodate insertion of Val‐β6. These results also strengthen our previous conclusion that β88 amino acid stereospecificity is more critical than that of β85 for insertion of β6 Val.
Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy
Authors: Slupsky Carolyn M. , Smillie Lawrence B. , Sykes Brian D. , Reinach Fernando C. 
Published: February 2009

AbstractThe solution secondary structure of calcium‐saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium‐saturated NTnC (residues 1–90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium‐saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium‐saturated TnC with the X‐ray crystal structure of half‐saturated TnC reveals differences in the ϕ/Ψ angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular ϕ/Ψ angles in the crystal structure, producing a kink in the B helix, whereas in calcium‐saturated TnC, Glu 41 has helical ϕ/Ψ angles, resulting in a straight B helix. The linker between the N and C domains of calcium‐saturated TnC is flexible in the solution structure.
Limited proteolysis of native proteins: The interaction between avidin and proteinase K
Authors: Ellison Derek , Beynon Robert J. , Hinton John , Hubbard Simon J. 
Published: February 2009

AbstractAvidin is a tetramer of 16‐kDa subunits that have a high affinity for biotin. Proteolysis of native apoavidin by proteinase K results in a limited attack at the loop between β‐strands 3 and 4, involving amino acids 38–43. Specifically, sites of proteolysis are at Thr 40‐Ser 41 and Asn 42‐Glu 43. The limited proteolysis results in an avidin product that remains otherwise intact and which has enhanced binding for 4′‐hydroxyazobenzene‐2‐benzoic acid (HABA), a chromogenic reporter that can occupy the biotin‐binding site. Saturation of the biotin‐binding site with the natural ligand protects avidin from proteolysis, but saturation with HABA enhances the rate of proteolysis of the same site.Analysis of the three‐dimensional structures of apoavidin and holoavidin reveals that the 3–4 loop is accessible to solvent and scores highly in an algorithm developed to identify sites of proteolytic attack. The structure of holoavidin is almost identical to the apoprotein. In particular, the 3–4 loop has the same structure in the apo and holo forms, yet there are marked differences in proteolytic susceptibility of this region. Evidence suggests that the 3–4 loop is rather mobile and flexible in the apoprotein, and that it becomes constrained upon ligand binding. In one crystal structure of the apoprotein, this loop appears constrained by contacts with symmetry‐related molecules. Structural analyses suggest that the “lid” to the biotin‐binding site, formed by the 3–4 loop, is displaced and made more accessible by HABA binding, thereby enhancing its proteolytic susceptibility.
Solvent accessibility as a predictive tool for the free energy of inhibitor binding to the HIV‐1 protease
Authors: Nauchitel Vladimir , Verde M. Carmen Villa , Sussman Fredy 
Published: February 2009

AbstractWe have developed a simple approach for the evaluation of the free energies of inhibitor binding to the protease of the human immunodeficiency virus (HIV‐1 PR). Our algorithm is based on the observation that most groups that line the binding pockets of this enzyme are hydrophobic in nature. Based on this fact, we have likened the binding of an inhibitor to this enzyme to its transfer from water to a medium of lower polarity. The resulting expression produced values for the free energy of binding of inhibitors to the HIV‐1 PR that are in good agreement with experimental values. The additive nature of this approach has enabled us to partition the free energy of binding into the contributions of single fragments. The resulting analysis clearly indicates the existence of a ranking in the participation of the enzyme's subsites in binding. Although all the enzyme's pockets contribute to binding, the ones that bind the P2‐P'2 span of the inhibitor are in general the most critical for high inhibitor potency. Moreover, our method has allowed us to determine the nature of the functional groups that fit into given enzyme binding pockets. Perusal of the energy contributions of single side chains has shown that a large number of hydrophobic and aromatic groups located in the central portion of the HIV‐1 PR inhibitors present optimal binding. All of these observations are in agreement with experimental evidence, providing a validation for the physical relevancy of our model.
The picornaviral 3C proteinases: Cysteine nucleophiles in serine proteinase folds
Authors: Malcolm Bruce A. 
Published: February 2009

AbstractThe 3C proteinases are a novel group of cysteine proteinases with a serine proteinase‐like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X‐ray crystallography opens the door for the application of the latest advances in computer‐assisted identification and design of anti‐proteinase therapeutic/chemoprophylactic agents.
Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A
Authors: Oblatt‐Montal Myrta , Yamazaki Masahito , Nelson Richard , Montal Mauricio 
Published: February 2009

AbstractSynthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel‐forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation‐selective channels when reconstituted in planar lipid bilayers. As predicted, the self‐assembled conductive oligomers express heterogeneous single‐channel conductances. The most frequent openings exhibit single‐channel conductance of 12 and 7 pS in 0.5 M NaC1, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic α‐helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel‐forming activity of intact botulinum toxin A.
C‐terminal specific protein degradation: Activity and substrate specificity of the Tsp protease
Authors: Keiler Kenneth C. , Silber Karen R. , Sauer Robert T. , Downard Kevin M. , Papayannopoulos Ioannis A. , Biemann Klaus 
Published: February 2009

AbstractThe activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free α‐carboxylate at the C‐terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C‐terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C‐terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.
Kinetics of interaction of partially folded proteins with a hydrophobic dye: Evidence that molten globule character is maximal in early folding intermediates
Authors: Engelhard Markus , Evans Philip A. 
Published: February 2009

AbstractInteraction with 8‐anilino‐1‐naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates.Many of these complications can be avoided by experiments where the ANS is introduced only after folding has been allowed to proceed for a variable time. The initial fluorescence intensity after mixing, resulting only from rapid and therefore hopefully relatively nonperturbing interactions with the protein, can be monitored at different refolding times to provide a better reflection of the progress of the reaction, uncomplicated by dye interaction effects. Such studies of the folding of carbonic anhydrase and α‐lactalbumin have been compared with conventional single‐mix experiments and large discrepancies observed. When ANS was present throughout refolding, time‐dependent changes attributed to the formation or reorganization of protein‐ANS complexes were clearly superimposed on those associated with the actual progress of refolding, and the folding kinetics and population of intermediates were also substantially perturbed by the dye. Thus, it is clear that the pulse method, though cumbersome, should be used where refolding reactions are to be probed by dye binding.The results emphasize that fluorescence enhancement tends to be greatest in early intermediates, in contrast to what, for carbonic anhydrase at least, might appear to be the case from the more conventional experiments. Later intermediates in the folding of both of these proteins actually induce little fluorescence enhancement and therefore may be quite different in nature from equilibrium molten globule states.
Characterization of leucine zipper complexes by electrospray ionization mass spectrometry
Authors: Wendt Hans , Dürr Eberhard , Thomas Richard M. , Przybylski Michael , Bosshard Hans Rudolf 
Published: February 2009

AbstractThe development of “soft” ionization methods has enabled the mass spectrometric analysis of higher‐order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI‐MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI‐MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled‐coil structures. ESI‐MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4‐pl of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac‐EYEALEKKLAAX1EAKX2QALEKKLEALEHG‐amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI‐MS as could the dimeric nature of GCN4‐pl. The formation of heteromeric two‐ and three‐stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4‐pl and LZ, was demonstrated by ESI‐MS and confirmed by fluorescence quenching experiments on fluorescein‐labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled‐coil domains.
Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy
Authors: Pountney Dean L. , Henehan Colin J. , Vasak Milan 
Published: February 2009

AbstractFar‐UV CD, 1H‐NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well‐characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 μg total protein) and, unlike either CD or 1H‐NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.
The distribution of α‐helix propensity along the polypeptide chain is not conserved in proteins from the same family
Authors: Muñoz Victor , Blanco Francisco J. , Serrano Luis 
Published: February 2009

AbstractWe address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of α‐helices. We have analyzed by CD the conformation of peptides corresponding to the five α‐helices of two α/β parallel proteins (ComA and Ara). The large α‐helical population of peptide ComA‐4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21‐ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275–296). Comparison of the helical content of equivalent peptides reveals that protein α‐helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.
Predicting oligomerization states of coiled coils
Authors: Woolfson Derek N. , Alber Tom 
Published: February 2009

AbstractAn algorithm based on the profile method was developed that faithfully distinguishes between the amino acid sequences of dimeric and trimeric coiled coils. Normalized sequence profiles derived from nonhomologous, two‐and three‐stranded, coiled‐coil sequences with unambiguous registers were used to assign dimer and trimer propensities to test sequences. The difference between the dimer and trimer profile scores accurately reflected the preferred oligomerization state. The method relied on two strategies that may be generally applicable to profile calculations —profile values of solvent‐exposed residues and of amino acids that were underrepresented in the database were given zero weight. Differences between the dimer and trimer profiles revealed sequence patterns that match and extend experimental studies of oligomer specification.
Purification and preliminary X‐ray crystallographic studies of recombinant L‐ribulose‐5‐phosphate 4‐epimerase from escherichia coli
Authors: Andersson Arnold , Schneider Gunter , Lindqvist Ylva 
Published: February 2009

AbstractThe araD gene from Escherichia coli, coding for L‐ribulose‐5‐phosphate 4‐epimerase, was overexpressed and the resulting enzyme was purified to homogeneity. Crystals of L‐ribuIose‐5‐phosphate 4‐epimerase, obtained with 4.0 M sodium formate as precipitant, belong to space group P4212 with unit cell dimensions a = b = 107.8 Å and c = 281.4 Å and diffract to at least 2.2 Å resolution. Density measurements of these crystals are consistent with eight subunits in the asymmetric unit.
Crystallization and preliminary X‐ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor
Authors: Keefe Lisa J. , Ginell Stephan L. , Westbrook Edwin M. , Anderson Carl W. 
Published: February 2009

AbstractRecombinant human adenovirus serotype 2 proteinase (both native and selenomethionine‐substituted) has been crystallized in the presence of the serotype 12, 11‐residue peptide cofactor. The crystals (space group P3121 or P3221, one molecule per asymmetric unit, a = b = 41.3 Å, c = 197.0 Å) grew in solutions containing 20–40% 2‐methyl‐2,4‐pentanediol (MPD), 0.1–0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0–7.5. Diffraction data (84% complete to 2.2 Å resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 × 1,024 pixel array) charge‐coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine‐substituted proteinase, 65% complete to 2.0 Å resolution with Rmerge values ranging 0.05–0.07, have been collected at three X‐ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.
Structural similarity between the pleckstrin homology domain and verotoxin: The problem of measuring and evaluating structural similarity
Authors: Orengo C.A. , Swindells M.B. , Michie A.D. , Zvelebil M.J. , Driscoll P.C. , Waterfield M.D. , Thornton J.M. 
Published: February 2009

AbstractAn unexpected structural similarity is described between the pleckstrin homology (PH) domain and verotoxin. This similarity has escaped detection primarily due to the differences in topology that exist between the two proteins. By comparing this result with two previously reported similarities for the PH domain, one with the lipocalins and another with the FK506 binding protein, we discuss the problems of measuring and assessing structural similarities.
Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain
Authors: Helms Larry R. , Wetzel Ronald 
Published: February 2009

AbstractIt is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.
Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues
Authors: Colette Daubner S. , Ehrlich Joel I. , Ramsey Andrew J. , Fitzpatrick Paul F. 
Published: February 2009

AbstractTyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild‐type enzyme, about 0.6 atoms/sub‐unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild‐type enzyme, and slightly altered V/Km values for both tyrosine and 6‐methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild‐type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.