bluuebunny/crossref_metadata_2025_split huggingface dataset:
Random dissection to select for protein split sites and its application in protein fragment complementation
Authors: Chen Yong , Li Shuang , Chen Tingjian , Hua Hui , Lin Zhanglin 
Published: February 2009

AbstractTo identify protein split sites quickly, a selection procedure by using chloramphenicol acetyl transferase (CAT) as reporter was introduced to search for folded protein fragments from libraries generated by random digestion and reassembly of the target gene, which yielded an abundant amount of DNA fragments with controllable lengths. Experimental results of tryptophan synthase alpha subunit (TSα) and TEM‐1 β‐lactamase agreed well with what the literature has reported. The solubility of these fragments correlated roughly with the minimum inhibitory concentrations of the CAT fusions. The application of this dissection protocol to protein fragment complementation assay (PCA) was evaluated using aminoglycoside‐3′‐phosphotransferase I (APH(3′)‐I) as a model protein. Three nearly bisectional sites and a number of possible split points were identified, and guided by this result, four novel pairs of fragments were tested for complementation. Three out of four pairs partially restored the APH activity with the help of leucine zippers, and a truncated but active APH(3′)‐I (Δ1–25) was also found. Finally, the weakly active APH(3′)‐I‐(1–253)NZ/CZ (254–271) containing a short 18 residue tag was further improved by error‐prone PCR, and a best mutant was obtained showing a fourfold improvement after just one round of evolution. These results demonstrate that protein random dissection based on the CAT selection can provide an efficient search for protein breakage points and guide the design of fragments for protein complementation assay. Furthermore, more active fragment pairs can be achieved with the classical directed evolution approach.
Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs
Authors: Risso Valeria A. , Primo María E. , Ermácora Mario R. 
Published: February 2009

AbstractB. licheniformis exo‐small β‐lactamase (ESBL) has a complex architecture with twelve α helices and a five‐stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL α helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.
X‐ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus
Authors: Day Michael W. , Hsu Barbara T. , Joshua‐Tor Leemor , Park Jae‐Bum , Zhou Zhi Hao , Adams Michael W. W. , Rees Douglas C. 
Published: February 2009

AbstractThe structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 °C, have been determined by X‐ray crystallography to a resolution of 1.8 å. Crystals of this rubredoxin grow in space group P212121 with room temperature cell dimensions a = 34.6 å, b = 35.5 å, and c = 44.4 å. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X‐PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 å and 2.06°, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 å and in bond angles of 1.95°. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen‐bonding network in the β‐sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino‐terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.
Redox properties of protein disulfide isomerase (dsba) from escherichia coli
Authors: Wunderlich Martina , Glockshuber Rudi 
Published: February 2009

AbstractThe redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 × 10−4 M was determined, corresponding to a standard redox potential (E'0) of DsbA of ‐0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 × 10−3 M to 3.9 × 10−5 M between pH 4 and 8.5. The redox state‐dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase “at work” during catalysis of oxidative protein folding.
Estimation of the maximum change in stability of globular proteins upon mutation of a hydrophobic residue to another of smaller size
Authors: Lee B. 
Published: February 2009

AbstractAlthough the hydrophobic effect is generally considered to be one of the most important forces in stabilizing the folded structure of a globular protein molecule, there is a lack of consensus on the precise magnitude of this effect. The magnitude of the hydrophobic effect is most directly measured by observing the change in stability of a protein molecule when an internal hydrophobic residue is mutated to another of smaller size. Results of such measurements have, however, been confusing because they vary greatly and are generally considerably larger than expected from the transfer free energies of corresponding small molecules. In this article, a thermodynamic argument is presented to show (1) that the variation is mainly due to that in the flexibility of the protein molecule at the site of mutation, (2) that the maximum destabilization occurs when the protein at the site of mutation is rigid, in which case the value of the destabilization is approximately given by the work of cavity formation in water, and (3) that the transfer free energy approximately gives the minimum of the range of variations. The best numerical agreements between the small molecule and the protein systems are obtained when the data from the small molecule system are expressed as the molarity‐based standard free energies without other corrections.
Electrostatic stabilization in four‐helix bundle proteins
Authors: Robinson Clifford R. , Sligar Stephen G. 
Published: February 2009

AbstractCharge substitutions generated by site‐directed mutagenesis at the termini of adjacent anti‐parallel α‐helices in a four‐helix bundle protein were used to determine a precise value for the contribution of indirect charge‐charge interactions to overall protein stability, and to simulate the electrostatic effects of α‐helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti‐parallel helices in the four‐helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four‐helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti‐parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti‐parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.
NMR analysis of staphylococcal nuclease thermal quench refolding kinetics
Authors: Kautz Roger A. , Fox Robert O. 
Published: February 2009

AbstractThermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 °C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116‐Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis‐trans equilibrium. The rate of trans → cis isomerization in the nativelike nuclease intermediate is approximately 100‐fold faster than that observed in a Lys‐Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116‐Pro 117 peptide bond is comparable to that observed for other X‐Pro isomerizations.
Representing an ensemble of NMR‐derived protein structures by a single structure
Authors: Sutcliffe Michael J. 
Published: February 2009

AbstractThe usefulness of representing an ensemble of NMR‐derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of ϕΨ torsion angles and the distribution of χ1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the “average” of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the “best” rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue.
ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway
Authors: Craig Douglas B. , Wallace CARMICHAEL J. A. 
Published: February 2009

AbstractEukaryotic cytochrome c possesses an ATP‐binding site of substantial specificity and high affinity that is conserved between highly divergent species and which includes the invariant residue arginine91. Such evolutionary conservatism strongly suggests a physiological role for ATP binding that demands further investigation. We report the preparation of adducts of the protein and the affinity labels 8‐azido adenosine 5′‐triphosphate, adenosine 5′‐triphosphate‐2′, 3′‐dialdehyde, and 5′‐p‐fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine91‐containing site, yet the reaction of the latter, although specific, occurred elsewhere, suggesting caution is necessary in its use. None of the adducts displayed significant modification of global structure, stability, or physicochemical properties, leading us to believe that the 8‐N3‐ATP and oATP adducts are good stabilized models of the noncovalent interaction; yet modification led to significant, and sometimes pronounced, effects on biological activity. We therefore propose that the role of ATP binding to this site, which we have shown to occur when the phosphorylation potential of the system is high under the equivalent of physiological conditions, is to cause a decrease in electron flow through the mitochondrial electron transport chain. Differences in the degree of inhibition produced by differences in adduct chemistry suggest that this putative regulatory role is mediated primarily by electrostatic effects.
Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPβ, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors
Authors: Cho Hyeongjin , Krishnaraj Ravichandran , Walsh Christopher T. , Itoh Michyasu , Saito Haruo , Kitas Eric , Bannwarth Willi 
Published: February 2009

AbstractThe transmembrane PTPase HPTPβ differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354‐amino acid, 41‐kDa human PTPβ catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTPβ shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9‐11 residues from 1ck, src, and PLCγ with Km values of 2, 4, and 1 μM, some 40‐200‐fold lower than the other two PTPases. With kcat values of 30‐205 s−1, the catalytic efficiency, kcat/Km, of the HPTPβ 41‐kDa catalytic domain is very high, up to 5.7 × 107 M−1 s−1. The peptides corresponding to PLCγ (766‐776) and EGFR (1, 167‐1, 177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTPβ catalytic domain. While exchange of the alanine residue at the +2 position of the PLCγ (Km of 1 μM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35‐fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 μM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the ‐2 position of the EGFR peptide decreased the Km value 34‐fold to 3 μM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.
Site‐directed mutagenesis of Klebsiella aerogenes urease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis
Authors: Park Il‐Seon , Hausinger Robert P. 
Published: February 2009

AbstractComparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the γ subunit, H39 and H41 in β, and H134, H136, H219, H246, H312, H320, and H321 in the α subunit of the Klebsiella aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site‐directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The γH96A, βH39A, βH41A, αH312A, and αH321A mutant proteins possess activities and nickel contents similar to wild‐type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the αH134A, αH136A, and αH246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild‐type enzyme. These results are consistent with αH134, αH136, and αH246 functioning as nickel ligands. The αH219A protein is active and has nickel (∼1.9% and ∼80%, respectively, when compared to wild‐type protein) but exhibits a very high Km value (1, 100 + 40 mM compared to 2.3 + 0.2 mM for the wild‐type enzyme). These results are compatible with αH219 having some role in facilitating substrate binding. Finally, the αH320A protein (Km = 8.3 + 0.2 mM) only displays ∼0.003% of the wild‐type enzyme activity, despite having a normal nickel content. Unlike the wild‐type and αH219A ureases, this mutant protein was not inactivated by diethylpyrocarbonate (DEP), consistent with αH320 being the DEP‐reactive general base that facilitates catalysis.
Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis
Authors: Hu James C. , Sauer Robert T. , Newell Nicholas E. , Tidor Bruce 
Published: February 2009

AbstractCombinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short α‐helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiae transcription factor GCN4 were randomized to these four residues in a λ repressor–leucine zipper fusion protein, resulting in 65, 536 possible residue combinations. Roughly three‐fourths of these combinations allowed stable coiled‐coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and non‐functional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix‐forming propensity and favorable intrasubunit and intersubunit charge‐charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.
Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry
Authors: Baca Manuel , Kent Stephen B. H. , Alewood Paul F. 
Published: February 2009

AbstractAn important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV‐1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' β‐turn mimic of predetermined three‐dimensional structure. The mimic β‐turn replaced residues Gly16, 17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry‐related type I' β‐turns in the native, unliganded enzyme. The β‐turn mimic‐containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16, 17‐containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the β‐turn at residues 15–18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid β‐turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a “molecular kit set” approach to protein design and synthesis.
Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore
Authors: Van Beeumen Jozef J. , Devreese Bart V. , Van Bun Stefaan M. , Hoff Wouter D. , Hellingwerf Klaas J. , Meyer Terrance E. , Cusanovich Michael A. , Mcree Duncan E. 
Published: February 2009

AbstractThe complete amino acid sequence of the 125‐residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to beMEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV.This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4Åresolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533‐6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%).The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light‐induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein.The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore‐binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+ 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.