bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogenetic and genetic variation among Fe(II)-oxidizing acidithiobacilli supports the view that these comprise multiple species with different ferrous iron oxidation pathways
Authors: Amouric Agnès , Brochier-Armanet Céline , Johnson D. Barrie , Bonnefoy Violaine , Hallberg Kevin B.
Published: October 2010

Autotrophic acidophilic iron- and sulfur-oxidizing bacteria of the genus Acidithiobacillus constitute a heterogeneous taxon encompassing a high degree of diversity at the phylogenetic and genetic levels, though currently only two species are recognized (Acidithiobacillus ferrooxidans and Acidithiobacillus ferrivorans). One of the major functional disparities concerns the biochemical mechanisms of iron and sulfur oxidation, with discrepancies reported in the literature concerning the genes and proteins involved in these processes. These include two types of high-potential iron–sulfur proteins (HiPIPs): (i) Iro, which has been described as the iron oxidase; and (ii) Hip, which has been proposed to be involved in the electron transfer between sulfur compounds and oxygen. In addition, two rusticyanins have been described: (i) rusticyanin A, encoded by the rusA gene and belonging to the well-characterized rus operon, which plays a central role in the iron respiratory chain; and (ii) rusticyanin B, a protein to which no function has yet been ascribed. Data from a multilocus sequence analysis of 21 strains of Fe(II)-oxidizing acidithiobacilli obtained from public and private collections using five phylogenetic markers showed that these strains could be divided into four monophyletic groups. These divisions correlated not only with levels of genomic DNA hybridization and phenotypic differences among the strains, but also with the types of rusticyanin and HiPIPs that they harbour. Taken together, the data indicate that Fe(II)-oxidizing acidithiobacilli comprise at least four distinct taxa, all of which are able to oxidize both ferrous iron and sulfur, and suggest that different iron oxidation pathways have evolved in these closely related bacteria.
Heterochromatin protein 1 (HP1) connects the FACT histone chaperone complex to the phosphorylated CTD of RNA polymerase II
Authors: Kwon So Hee , Florens Laurence , Swanson Selene K. , Washburn Michael P. , Abmayr Susan M. , Workman Jerry L.
Published: October 2010

Heterochromatin protein 1 (HP1) is well known as a silencing protein found at pericentric heterochromatin. Most eukaryotes have at least three isoforms of HP1 that play differential roles in heterochromatin and euchromatin. In addition to its role in heterochromatin, HP1 proteins have been shown to function in transcription elongation. To gain insights into the transcription functions of HP1, we sought to identify novel HP1-interacting proteins. Biochemical and proteomic approaches revealed that HP1 interacts with the histone chaperone complex FACT (facilitates chromatin transcription). HP1c interacts with the SSRP1 (structure-specific recognition protein 1) subunit and the intact FACT complex. Moreover, HP1c guides the recruitment of FACT to active genes and links FACT to active forms of RNA polymerase II. The absence of HP1c partially impairs the recruitment of FACT into heat-shock loci and causes a defect in heat-shock gene expression. Thus, HP1c functions to recruit the FACT complex to RNA polymerase II.
PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas
Authors: Ozawa Tatsuya , Brennan Cameron W. , Wang Lu , Squatrito Massimo , Sasayama Takashi , Nakada Mitsutoshi , Huse Jason T. , Pedraza Alicia , Utsuki Satoshi , Yasui Yoshie , Tandon Adesh , Fomchenko Elena I. , Oka Hidehiro , Levine Ross L. , Fujii Kiyotaka , Ladanyi Marc , Holland Eric C.
Published: October 2010

Gene rearrangement in the form of an intragenic deletion is the primary mechanism of oncogenic mutation of the epidermal growth factor receptor (EGFR) gene in gliomas. However, the incidence of platelet-derived growth factor receptor-α (PDGFRA) gene rearrangement in these tumors is unknown. We investigated the PDGFRA locus in PDGFRA-amplified gliomas and identified two rearrangements, including the first case of a gene fusion between kinase insert domain receptor (KDR) (VEGFRII) and the PDGFRA gene, and six cases of PDGFRAΔ8, 9, an intragenic deletion rearrangement. The PDGFRAΔ8, 9 mutant was common, being present in 40% of the glioblastoma multiformes (GBMs) with PDGFRA amplification. Tumors with these two types of PDGFRA rearrangement displayed histologic features of oligodendroglioma, and the gene products of both rearrangements showed constitutively elevated tyrosine kinase activity and transforming potential that was reversed by PDGFR blockade. These results suggest the possibility that these PDGFRA mutants behave as oncogenes in this subset of gliomas, and that the prevalence of such rearrangements may have been considerably underestimated.
Aedes aegypti: An Emerging Model for Vector Mosquito Development: Figure 1.
Authors: Clemons Anthony , Haugen Morgan , Flannery Ellen , Tomchaney Michael , Kast Kristopher , Jacowski Caitlin , Le Christy , Mori Akio , Simanton Holland Wendy , Sarro Joseph , Severson David W. , Duman-Scheel Molly
Published: October 2010

INTRODUCTION

Blood-feeding mosquitoes, including the dengue and yellow fever vectorAedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although theAe. aegyptigenome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development inAe. aegyptior other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses ofAe. aegyptias a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes duringAe. aegyptidevelopment. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.
Stable Producer Cell Lines for Adeno-Associated Virus (AAV) Assembly
Authors: Chadeuf Gilliane , Salvetti Anna
Published: October 2010

INTRODUCTION

Stable producer cell lines containing both the rep and cap genes and recombinant adeno-associated virus (rAAV) vectors can be infected with a helper virus to provide reliable and efficient production of rAAV stocks. However, the development of these cell lines is time-consuming. The procedure described here is therefore recommended only for studies requiring the production of high amounts of rAAV, such as preclinical studies performed in large animals.
Imaging Collagen II Using Atomic Force Microscopy (AFM)
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli
Published: October 2010

INTRODUCTION

Collagen II is a fibrous protein that assembles from basic tropocollagen subunits to form extracellular supramolecular fiber networks within cartilage tissue. Tropocollagen subunits of ~300 nm in length self-assemble first into pentameric uniform microfibrils, which fuse into bigger collagen fibrils that can range from 10 nm to 500 nm in diameter. The collagen fibrils display a characteristic 67-nm repeat because of the staggering of individual collagen molecules with respect to each other. This protocol demonstrates how to prepare collagen protein samples for analysis by atomic force microscopy (AFM). It also describes the steps for generating AFM images of collagen samples during and after manipulation to analyze collagen self-assembly.
Efficient Genomic DNA Extraction from Low Target Concentration Bacterial Cultures Using SCODA DNA Extraction Technology
Authors: So Austin , Pel Joel , Rajan Sweta , Marziali Andre
Published: October 2010

INTRODUCTION

Methods for the extraction of nucleic acids are straightforward in instances where there is ample nucleic acid mass in the sample and contamination is minimal. However, applications in areas such as metagenomics, life science research, clinical research, and forensics, that are limited by smaller amounts of starting materials or more dilute samples, require sample preparation methods that are more efficient at extracting nucleic acids. Synchronous coefficient of drag alteration (SCODA) is a novel electrophoretic nucleic acid purification technology that has been tested successfully with both highly contaminated and dilute samples and is a promising candidate for new sample preparation challenges. In this article, as an example of SCODA’s performance with limited sample material, we outline a genomic DNA (gDNA) extraction protocol from low abundance cultures of Escherichia coli DH10B. This method is equally well suited to high biomass samples.
Culturing and Egg Collection of Aedes aegypti
Authors: Clemons Anthony , Mori Akio , Haugen Morgan , Severson David W. , Duman-Scheel Molly
Published: October 2010

INTRODUCTION

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.
Whole-Mount In Situ Hybridization for Analysis of Gene Expression during Aedes aegypti Development: Figure 1.
Authors: Haugen Morgan , Tomchaney Michael , Kast Kristopher , Flannery Ellen , Clemons Anthony , Jacowski Caitlin , Simanton Holland Wendy , Le Christy , Severson David , Duman-Scheel Molly
Published: October 2010

INTRODUCTION

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.
Immunohistochemical Analysis of Protein Expression during Aedes aegypti Development: Figure 1.
Authors: Clemons Anthony , Flannery Ellen , Kast Kristopher , Severson David , Duman-Scheel Molly
Published: October 2010

INTRODUCTION

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and gene products s that regulate development are of particular interest. This protocol for immunohistochemical analysis of protein expression can be used to analyze expression of developmental proteins of interest in Ae. aegypti embryos, larvae, and pupae, which will be critical for the development of markers for particular developing tissues.
Atomic Force Microscopy for Biological Imaging and Mechanical Testing across Length Scales
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli
Published: October 2010

INTRODUCTION

Atomic force microscopy (AFM) offers researchers a unique opportunity to visualize, manipulate, and quantitatively assess structural and mechanical aspects of native biological samples with nanometer resolution. An unparalleled advantage of AFM over other high-resolution microscopes is that biological specimens, ranging from tissues to cells to molecules, can be investigated in physiologically relevant aqueous environments. The AFM can be operated at 37°C, which makes it ideal for in situ cell or tissue studies. Combining an optical microscope with an AFM makes it possible to directly correlate structural/nanomechanical changes with optical/fluorescence images. This ability to simultaneously acquire structural and function information is unprecedented in biology. This article introduces the basics of AFM for imaging and investigating the properties of biological samples.
The Arabidopsis C3H2C3-Type RING E3 Ubiquitin Ligase AtAIRP1 Is a Positive Regulator of an Abscisic Acid-Dependent Response to Drought Stress
Authors: Ryu Moon Young , Cho Seok Keun , Kim Woo Taek
Published: October 2010

Ubiquitination is a eukaryotic posttranslational protein modification that is mediated by the cascade of E1, E2, and E3 ubiquitin (Ub) ligases and is involved in regulating numerous cellular functions. In this study, we obtained 100 different Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant plants in which RING E3 Ub ligase genes were suppressed and monitored their phenotypes in the presence of exogenous abscisic acid (ABA), a plant stress hormone. One of these loss-of-function mutants displayed ABA-insensitive phenotypes at the germination stage and was named atairp1 (for Arabidopsis ABA-insensitive RING protein 1). AtAIRP1 encodes a cytosolic protein containing a single C3H2C3-type RING motif with in vitro E3 Ub ligase activity. AtAIRP1 was significantly induced by ABA and drought stress. In contrast to atairp1 mutant plants, AtAIRP1-overexpressing transgenic plants (35S:AtAIRP1-sGFP) were hypersensitive to exogenous ABA in terms of radicle emergence, cotyledon development, root elongation, and stomatal closure. Ectopic expression of AtAIRP1-sGFP in atairp1 effectively rescued the loss-of-function ABA-insensitive phenotype. Both 35S:AtAIRP1-sGFP and atairp1/35S:AtAIRP1-sGFP plants accumulated higher amounts of hydrogen peroxide in response to exogenous ABA than did wild-type and atairp1 mutant plants. AtAIRP1 overexpressors were markedly tolerant to severe drought stress, as opposed to atairp1, which was highly susceptible. The levels of drought stress-related genes and basic leucine zipper transcription factor genes were up-regulated in the 35S:AtAIRP1-sGFP lines relative to wild-type and atairp1 mutant plants in response to ABA. Overall, these results suggest that AtAIRP1, a C3H2C3-type RING E3 Ub ligase, is a positive regulator in the Arabidopsis ABA-dependent drought response.
HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis
Authors: Nowara Daniela , Gay Alexandra , Lacomme Christophe , Shaw Jane , Ridout Christopher , Douchkov Dimitar , Hensel Götz , Kumlehn Jochen , Schweizer Patrick
Published: October 2010

Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.
Identification of tissue transglutaminase‐reactive lysine residues in glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Orru Stefania , Ruoppolo Margherita , Francese Simona , Vitagliano Luigi , Marino Gennaro , Esposito Carla
Published: October 2010

Polyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross‐links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine‐containing peptide was used as probe for labeling the amino‐donor sites. SDS gel electrophoresis of a time‐course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino‐donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino‐donor residues modified by tissue transglutaminase.
Unique oligomeric intermediates of bovine liver catalase
Authors: Prakash Koodathingal , Prajapati Shashi , Ahmad Atta , Jain S.K. , Bhakuni Vinod
Published: October 2010

Catalases, although synthesized from single genes and built up from only one type of subunit, exist in heterogeneous form with respect to their conformations and association states in biological systems. This heterogeneity is not of genetic origin, but rather reflects the instability of this oligomeric heme enzyme. To understand better the factors that stabilize the various association states of catalase, we performed studies on the multimeric intermediates that are stabilized during guanidine‐hydrochloride‐ and urea‐induced unfolding of bovine liver catalase (BLC). For the first time, we have observed an enzymatically active, folded dimer of native BLC. This dimer has slightly higher enzymatic activity and altered structural properties compared to the native tetramer. Comparative studies of the effect of NaCl, GdmCl, and urea on BLC show that cation binding to negatively charged groups present in amino acid side chains of the enzyme leads to stabilization of an enzymatically active, folded dimer of BLC. Besides the folded dimer, an enzymatically active expanded tetramer and a partially unfolded, enzymatically inactive dimer of BLC were also observed. A complete recovery of native enzyme was observed on refolding of expanded tetramers and folded dimers; however, a very low recovery (maximum of ∼5%) of native enzyme was observed on refolding of partially unfolded dimers and fully unfolded monomers.
Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange
Authors: Walkenhorst William F. , Edwards Jason A. , Markley John L. , Roder Heinrich
Published: October 2010

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid‐denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly15N‐labeled H124L SNase was monitored as a function of refolding time by heteronuclear two‐dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen‐bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the β‐barrel and to a lesser degree in the α‐helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a β‐hairpin formed by strands 2 and 3 of the β‐barrel domain and a single site near the C‐terminus. The results indicate that formation of stable hydrogen‐bonded structure in a core region of the β‐sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.
Composites of local structure propensities: Evidence for local encoding of long‐range structure
Authors: Shortle David
Published: October 2010

To estimate how extensively the ensemble of denatured‐state conformations is constrained by local side‐chain–backbone interactions, propensities of each of the 20 amino acids to occur in mono‐ and dipeptides mapped to discrete regions of the Ramachandran map are computed from proteins of known structure. In addition, propensities are computed for the trans, gauche−, and gauche+ rotamers, with or without consideration of the values of phi and psi. These propensities are used in scoring functions for fragment threading, which estimates the energetic favorability of fragments of protein sequence to adopt the native conformation as opposed to hundreds of thousands of incorrect conformations. As finer subdivisions of the Ramachandran plot, neighboring residue phi/psi angles, and rotamers are incorporated, scoring functions become better at ranking the native conformation as the most favorable. With the best composite propensity function, the native structure can be distinguished from 300,000 incorrect structures for 71% of the 2130 arbitrary protein segments of length 40, 48% of 2247 segments of length 30, and 20% of 2368 segments of length 20. A majority of fragments of length 30–40 are estimated to be folded into the native conformation a substantial fraction of the time. These data suggest that the variations observed in amino acid frequencies in different phi/psi/chi1 environments in folded proteins reflect energetically important local side‐chain–backbone interactions, interactions that may severely restrict the ensemble of conformations populated in the denatured state to a relatively small subset with nativelike structure.
Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta
Authors: Lauble Hanspeter , Miehlich Burkhard , Förster Siegfried , Kobler Christoph , Wajant Harald , Effenberger Franz
Published: October 2010

Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL‐W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild‐type MeHNL and MeHNL‐W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL‐W128A for the unnatural substrates mandelonitrile and 4‐hydroxymandelonitrile are increased 9‐fold and ∼450‐fold, respectively, compared with the wild‐type MeHNL. The crystal structure of the MeHNL‐W128A substrate‐free form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active‐site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL‐W128A–4‐hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.
On the non‐respect of the thermodynamic cycle by DsbA variants
Authors: Moutiez Mireille , Quéméneur Eric , Burova Tatiana V. , Haertlé Thomas
Published: October 2010

The mechanism of the disulfide‐bond forming enzyme DsbA depends on the very low pKa of a cysteine residue in its active‐site and on the relative instability of the oxidized enzyme compared to the reduced one. A thermodynamic cycle has been used to correlate its redox properties to the difference in the free energies of folding (ΔΔGred/ox) of the oxidized and reduced forms. However, the relation was proved unsatisfied for a number of DsbA variants. In this study, we investigate the thermodynamic and redox properties of a highly destabilized variant DsbAP151A (substitution of cis‐Pro151 by an alanine) by the means of intrinsic tryptophan fluorescence and by high‐sensitivity differential scanning calorimetry (HS‐DSC). When the value of ΔΔGred/ox obtained fluorimetrically for DsbAP151A does not correlate with the value expected from its redox potential, the value of ΔΔGred/ox provided by HS‐DSC are in perfect agreement with the predicted thermodynamic cycle for both wild‐type and variant. HS‐DSC data indicate that oxidized wild‐type enzyme and the reduced forms of both wild‐type and variant unfold according to a two‐state mechanism. Oxidized DsbAP151A shows a deviation from two‐state behavior that implies the loss of interdomain cooperativity in DsbA caused by Pro151 substitution. The presence of chaotrope in fluorimetric measurements could facilitate domain uncoupling so that the fluorescence probe (Trp76) does not reflect the whole unfolding process of DsbAP151A anymore. Thus, theoretical thermodynamic cycle is respected when an appropriate method is applied to DsbA unfolding under conditions in which protein domains still conserve their cooperativity.
Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between β‐galactosidase and other glycohydrolases
Authors: Juers Douglas H. , Huber Reuben E. , Matthews Brian W.
Published: October 2010

β‐Galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an α/β barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an α/β barrel. Those that are most similar to the α/β barrel of E. coli β‐galactosidase have similar catalytic residues and belong to the so‐called “4/7 superfamily” of glycosyl hydrolases. The structure comparison suggests that β‐amylase should also be included in this family. Of three structure comparison methods tested, the “ProSup” procedure of Zu‐Kang and Sippl and the “Superimpose” procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli β‐galactosidase have topologies related to “jelly‐roll barrels” and “immunoglobulin constant” domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli β‐galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in β‐galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that β‐galactosidase arose from a prototypical single domain α/β barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.
The mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: Conformational flexibility and molecular chaperone activity
Authors: Yang Hongmei , Huang Sufang , Dai Hongzheng , Gong Yandao , Zheng Changxue , Chang Zengyi
Published: October 2010

Hsp16.3, the α‐crystallin‐related small heat shock protein of Mycobacterium tuberculosis that is maximally expressed during the stationary phase and is a major membrane protein, has been reported to form specific trimer‐of‐trimers structure and to act as an effective molecular chaperone (Chang Z et al., 1996, J Biol Chem 271:7218—7223). However, little is known about its action mechanism. In this study, Hsp16.3 conformational intermediates with dramatically increased chaperone activities were detected after treatment with very low concentrations of guanidine hydrochloride (0.05 M), urea (0.3 M), or mild heating (30 8C). The intermediates showed a significant increase in their capacity to bind the hydrophobic probe 1‐anilino‐8‐naphthalene sulfonate (ANS), indicating an increased exposure of hydrophobic surfaces. Interestingly, the greatest chaperone activities of Hsp16.3 were observed in the presence of 0.3 M guanidine HCl or when heated to 35 °C. CD spectroscopy studies revealed no significant changes in protein secondary and tertiary structures at these mild treatments. Our in vitro studies also indicate that long‐time‐heated Hsp16.3, heated even to temperatures as high as 85 °C, has almost the same, if not a slightly greater, chaperone activities as the native protein when cooled to room temperature and its secondary structures also almost recovered. Together, these results suggest that Hsp16.3 modulates its chaperone activity by exposing hydrophobic surfaces and that the protein structure is highly stable and flexible, thus highly adapted for its function.
The influence of C‐terminal extension on the structure of the “J‐domain” in E. coli DnaJ
Authors: Huang Kai , Flanagan John M. , Prestegard James H.
Published: October 2010

Two different recombinant constructs of the N‐terminal domain in Escherichia coli DnaJ were uniformly labeled with nitrogen‐15 and carbon‐13. One, DnaJ(1—78), contains the complete “J‐domain,” and the other, DnaJ(1—104), contains both the “J‐domain” and a conserved “G/F” extension at the C‐terminus. The three‐dimensional structures of these proteins have been determined by heteronuclear NMR experiments. In both proteins the “J‐domain” adopts a compact structure consisting of a helix‐turn‐helix‐loop‐helix‐turn‐helix motif. In contrast, the “G/F” region in DnaJ(1—104) does not fold into a well‐defined structure. Nevertheless, the “G/F” region has been found to have an effect on the packing of the helices in the “J‐domain” in DnaJ(1—104). Particularly, the interhelical angles between Helix IV and other helices are significantly different in the two structures. In addition, there are some local conformational changes in the loop region connecting the two central helices. These structural differences in the “J‐domain” in the presence of the “G/F” region may be related to the observation that DnaJ(1—78) is incapable of stimulating the ATPase activity of the molecular chaperone protein DnaK despite evidence that sites mediating the binding of DnaJ to DnaK are located in the 1—78 segment.
Stereoselectivity of mucorales lipases toward triradylglycerols—A simple solution to a complex problem
Authors: Scheib Holger , Pleiss Jürgen , Schmid Rolf D. , Kovac Andrea , Paltauf Fritz
Published: October 2010

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn‐2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'‐C1′ ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N′‐C1′ amide bond or a phenyl ring in sn‐2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn‐1 and sn‐3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn‐1. To explain these experimental observations, computer‐aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C‐terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn‐1 and sn‐3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.
The insect immune protein scolexin is a novel serine proteinase homolog
Authors: Finnerty Casey M. , Granados Robert R. , Karplus P. Andrew
Published: October 2010

Scolexin is a coagulation‐provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin‐like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.
For the record: Structural consequences of the active site substitution Cys181 → Ser in metallo‐β‐lactamase from bacteroides fragilis
Authors: Li Zhong , Herzberg Osnat , Rasmussen Beth A.
Published: October 2010

The metallo‐β‐lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of β‐lactam antibiotics. The crystal structure of the Zn2+‐bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the β‐lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo‐β‐lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 Å resolution. The overall structure of the mutant enzyme is the same as that of the wild‐type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild‐type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild‐type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.
Quench‐flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate
Authors: Tsui Vickie , Garcia Carlos , Cavagnero Silvia , Siuzdak Gary , Dyson H. Jane , Wright Peter E.
Published: October 2010

Folding of apomyoglobin is characterized by formation of a compact intermediate that contains substantial helicity. To determine whether this intermediate is obligatory or whether the protein can fold directly into the native state via an alternate parallel pathway, we have combined quench‐flow hydrogen‐exchange pulse labeling techniques with electrospray ionization mass spectrometry. The mass spectra of apomyoglobin obtained at various refolding times suggest that apomyoglobin indeed folds through a single pathway containing an obligatory intermediate with a significant hydrogen‐bonded secondary structure content.
Functional implications of structural differences between variants A and B of bovine β‐lactoglobulin
Authors: Qin Bin Y. , Jameson Geoffrey B. , Bewley Maria C. , Baker Edward N. , Creamer Lawrence K.
Published: October 2010

The structure of the trigonal crystal form of bovine β‐lactoglobulin variant B at pH 7.1 has been determined by X‐ray diffraction methods at a resolution of 2.22 Å and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine β‐lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014—14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61—67), there are small changes in main‐chain conformation, whereas the substitution V118A on β‐strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH‐dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.
Crystal structure of a designed, thermostable, heterotrimeric coiled coil
Authors: Nautiyal Shivani , Alber Tom
Published: October 2010

Electrostatic interactions are often critical for determining the specificity of protein‐protein complexes. To study the role of electrostatic interactions for assembly of helical bundles, we previously designed a thermostable, heterotrimeric coiled coil, ABC, in which charged residues were employed to drive preferential association of three distinct, 34‐residue helices. To investigate the basis for heterotrimer specificity, we have used multiwavelength anomalous diffraction (MAD) analysis to determine the 1.8 Å resolution crystal structure of ABC. The structure shows that ABC forms a heterotrimeric coiled coil with the intended arrangement of parallel chains. Over half of the ion pairs engineered to restrict helix associations were apparent in the experimental electron density map. As seen in other trimeric coiled coils, ABC displays acute knobs‐into‐holes packing and a buried anion coordinated by core polar amino acids. These interactions validate the design strategy and illustrate how packing and polar contacts determine structural uniqueness.
Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response innicotiana sylvestris
Authors: Toedt John M. , Braswell Emory H. , Schuster Todd M. , Yphantis David A. , Taraporewala Zenobia F. , Culver James N.
Published: October 2010

The hypersensitivity resistance response directed by the N' gene inNicotiana sylvestrisis elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild‐type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild‐type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4°C. Viral reconstitution kinetics at 20°C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20°C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allowsN. sylvestristo recognize the invading virus.
Crystal structure of wild‐type human procathepsin K
Authors: Sivaraman J. , Lalumière1 Manon , Ménard Robert , Cygler Miroslaw
Published: October 2010

Cathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild‐type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 å resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R‐factor of 0.194. The N‐terminal fragment of the proregion forms a globular domain while the C‐terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138–Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix α3p (67p–75p), which occupies the S' subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate.
Comparison of the backbone dynamics of the apo‐ and holo‐carboxy‐terminal domain of the biotin carboxy1 carrier subunit of Escherichia coli acety1‐CoA carboxylase
Authors: Yao Xiang , Soden Cylburn , Summers Michael F. , Beckett Dorothy
Published: October 2010

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl‐CoA carboxylase, a biotin‐dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein–protein interactions with three distinct partners, depending on its state of post‐translational modification. Apo‐BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post‐translational attachment of biotin to a single lysine residue on BCCP. Holo‐BCCP then interacts with the biotin carboxylase subunit of acetyl‐CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy‐biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl‐CoA to form malonyl‐CoA. The determinants of protein–protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C‐terminal domain fragment (residue 70–156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C‐terminal fragment (residue 77–156) of BCCP from Escherichia coli acetyl‐CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin‐binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein–protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin‐binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.
Tolerance of a protein to multiple polar‐to‐hydrophobic surface substitutions
Authors: Cordes Matthew H.J. , Sauer Robert T.
Published: October 2010

Hydrophobic substitutions at solvent‐exposed positions in two α‐helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild‐type Arc, with a rough correlation between the number of polar‐to‐hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar‐to‐hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a tm 6°C higher than wild‐type. By contrast, a helix A mutant with three similar polar‐to‐hydrophobic substitutions was 23°C less stable than wild‐type. Also, one set of three polar‐to‐hydrophobic substitutions in helix B was tolerated when introduced into the wild‐type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.
Topology and dynamics of the 10 kDa C‐terminal domain of DnaK in solution
Authors: Bertelsen Eric B. , Zhou Hongjun , Lowry David F. , Flynn Gregory C. , Dahlquist Frederick W.
Published: October 2010

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N‐terminal ATPase domain, a 17 kDa peptide‐binding domain, and a 10 kDa C‐terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C‐terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C‐terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high‐resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C‐terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full‐length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle‐like structure in solution.