bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein folding coupled to DNA binding in the catalytic domain of bacteriophage λ integrase detected by mass spectrometry
Authors: Kamadurai Hari B. , Subramaniam Srisunder , Jones R. Benjamin , Green‐Church Kari B. , Foster Mark P. 
Published: February 2003

AbstractBacteriophage λ integrase (λ‐Int) is the prototypical member of a large family of enzymes that catalyze site‐specific DNA recombination via single‐strand cleavage and the formation of a Holliday junction intermediate. Crystallographic and biochemical evidence indicate that substantial conformational change (i.e., folding) in the catalytic domain of the protein is required for substrate recognition and catalysis. We have examined the solution conformation of the catalytic domain (C170) in the absence and presence of a cognate “half‐site” DNA oligonucleotide by electrospray ionization mass spectrometry, and circular dichroism and fluorescence spectroscopy. The distribution of ions in the positive ion electrospray mass spectrum of the free protein reveals the presence of three distinct species in solution, one corresponding to the folded protein, one to the unfolded protein, and one to a dimer. In the presence of DNA, ions are observed only for the protein–DNA complex and the folded form of the free protein. We therefore conclude that DNA binding stabilizes the global fold of the protein in a manner that is consistent with folding‐coupled target recognition as a mechanism to control site‐specific recombination. Furthermore, we find that inspection of the charge state distribution of ions in electrospray mass spectra provides a quick and effective means to identify conformational heterogeneity of proteins in solution and to investigate dynamic protein–nucleic acid interactions.
Crystal structure of a defective folding protein
Authors: Saul Frederick A. , Mourez Michaël , Vulliez‐le Normand Brigitte , Sassoon Nathalie , Bentley Graham A. , Betton Jean‐Michel 
Published: February 2003

AbstractMaltose‐binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32→Asp and Ile 33→Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat‐shock periplasmic peptidyl‐prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose‐binding activity comparable with that of the wild‐type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose‐bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 Å resolution. The conformation of MalE31 closely resembles that of wild‐type MalE, with very small displacements of the mutated residues located in the loop connecting the first α‐helix to the first β‐strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild‐type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.
Equilibrium and kinetic studies on folding of canine milk lysozyme
Authors: Van Dael Herman , Haezebrouck Petra , Joniau Marcel 
Published: February 2003

AbstractThermal and chemical unfolding studies of the calcium‐binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped‐flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo‐canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s−1. Furthermore, the results show that, unlike its drastic effect on the stability, Ca2+‐binding only marginally affects the refolding kinetics. During the refolding process of apo‐CL non‐native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non‐native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.
A PDZ domain‐based assay for measuring HIV protease activity: Assay design considerations
Authors: Hamilton Aaron C. , Inglese James , Ferrer Marc 
Published: February 2003

AbstractWe have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain•peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ‐binding motif is exposed, and the product sequence bound specifically by a Eu3+chelate‐labeled GST–PDZ ([Eu3+]GST–PDZ). The practical applicability of this PDZ‐based detection method is determined by the affinity of the PDZ domain•peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ‐based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD‐95 was used, which preferentially recognizes the consensus sequence Ser‐X‐Val‐COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser‐X‐Leu‐COOH, can be used to extend the flexibility in the recognition of the carboxy‐terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.
Competing intrachain interactions regulate the formation of β‐sheet fibrils in bovine PrP peptides
Authors: Tahiri‐Alaoui Abdessamad , Bouchard Mario , Zurdo Jesús , James William 
Published: February 2003

AbstractAt the heart of the pathogenesis of transmissible spongiform encephalopathies (TSEs), such as BSE, scrapie, and Creutzfeldt–Jakob disease, lies a poorly understood structural rearrangement of PrP, an abundant glycoprotein of the nervous and lymphoid systems. The normal form (PrPC), rich in α‐helix, converts into an aberrant β‐sheet‐dominated form (PrPSc), which seems to be at the center of the pathotoxic symptoms observed in TSEs. To understand this process better at a molecular level, we have studied the interactions between different peptides derived from bovine PrP and their structural significance. We show that two unstructured peptides derived from the central region of bovine PrP, residues 115–133 and 140–152, respectively, interact stoichiometrically under physiological conditions to generate β‐sheet‐dominated fibrils. However, when both peptides are incubated in the presence of a third peptide derived from an adjoining α‐helical region (residues 153–169), the formation of β‐sheet‐rich fibrils is abolished. These data indicate that native PrPC helix 1 might inhibit the strong intrinsic β‐sheet‐forming propensity of sequences immediately N‐terminal to the globular core of PrPC, by keeping in place intrachain interactions that would prevent these amyloidogenic regions from triggering aggregation. Moreover, these results indicate new ways in which PrPSc formation could be prevented.
The effect of the polyproline II (PPII) conformation on the denatured state entropy
Authors: Ferreon Josephine C. , Hilser Vincent J. 
Published: February 2003

AbstractPolyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM‐5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard‐sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, ∼30% and ∼10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.
Two Nucleoside Uptake Systems in
Lactococcus lactis
: Competition between Purine Nucleosides and Cytidine Allows for Modulation of Intracellular Nucleotide Pools
Authors: Martinussen Jan , Wadskov-Hansen Steen L. L. , Hammer Karin 
Published: February 2003

ABSTRACT

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of
33
P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H
3
BO
3
-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in
Lactococcus lactis
were investigated by measuring the uptake of radioactively labeled nucleosides. The

K
m

for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.
Dual Role of Cysteine 172 in Redox Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity and Degradation
Authors: Marcus Yehouda , Altman-Gueta Hagit , Finkler Aliza , Gurevitz Michael 
Published: February 2003

ABSTRACT

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from
Synechocystis
sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.
Synergistic Effects of Cellulosomal Xylanase and Cellulases from
Clostridium cellulovorans
on Plant Cell Wall Degradation
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H. 
Published: February 2003

ABSTRACT

Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from
Clostridium cellulovorans
. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation. The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases. Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions. The possible mechanism of synergism between XynA and cellulases is discussed.
Genetic and Biochemical Characterization of the F-ATPase Operon from S
treptococcus sanguis
10904
Authors: Kuhnert Wendi L. , Quivey, Robert G. 
Published: February 2003

ABSTRACT

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species
Streptococcus sanguis
and
Streptococcus mutans
. Differences in the pH optima of the respective ATPases appears to be the main reason that
S. mutans
is more tolerant of low pH values than
S. sanguis
and hence pathogenic. We have recently reported the genetic arrangement for the
S. mutans
operon. For purposes of comparative structural biology we have also investigated the F-ATPase from
S. sanguis
. Here, we report the genetic characterization and expression in
Escherichia coli
of the
S. sanguis
ATPase operon. Sequence analysis showed a gene order of
atpEBFHAGDC
and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both
S. sanguis
and
S. mutans
; however, it is not induced to the same extent in the nonpathogenic
S. sanguis
. Expression studies with an
atpD
deletion strain of
E. coli
showed that
S. sanguis
-
E. coli
hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.
Efficient Turnover of Chlorocatechols Is Essential for Growth of
Ralstonia eutropha
JMP134(pJP4) in 3-Chlorobenzoic Acid
Authors: Pérez-Pantoja D. , Ledger T. , Pieper D. H. , González B. 
Published: February 2003

ABSTRACT

Ralstonia eutropha
JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters,

tfdC
I

D
I

E
I

F
I

and

tfdD
II

C
II

E
II

F
II

. Introduction of several copies of each gene cluster into
R. eutropha
JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the

tfdC
I

gene, and the derivative of strain JMP222 containing

tfdC
I

D
I

E
I

F
I

plus multiple copies of the

tfdC
I

gene could efficiently grow in 3-CB. In addition,

tfdC
I

and

tfdC
II

gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the
xylS
-
xylXYZL
genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.
Polymorphism in the Collagen-Like Region of the
Bacillus anthracis
BclA Protein Leads to Variation in Exosporium Filament Length
Authors: Sylvestre Patricia , Couture-Tosi Evelyne , Mock Michèle 
Published: February 2003

ABSTRACT

We recently identified a
Bacillus anthracis
glycoprotein which is a structural constituent of the exosporium filaments (P. Sylvestre, E. Couture-Tosi, and M. Mock, Mol. Microbiol.
45:
169-178, 2002). This
Bacillus
collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets. Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P. Le Flèche et al., BMC Microbiol.
1:
2, 2001), maps within the open reading frame encoding BclA. The
bclA
gene in 11
B. anthracis
strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames
bclA
gene sequence. The amino- and carboxy-terminal sequences surrounding the CLR are conserved. The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)
5
GDTGTT, named the BclA repeat. The length of the filament on the spore surface differed between the strains. We exchanged the
bclA
gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis. The length of the BclA CLR is responsible for the variation in filament length.
Exogenous Glutathione Completes the Defense against Oxidative Stress in
Haemophilus influenzae
Authors: Vergauwen Bjorn , Pauwels Frederik , Vaneechoutte Mario , Van Beeumen Jozef J. 
Published: February 2003

ABSTRACT

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus
Haemophilus
appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (γ-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the
Haemophilus influenzae
Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable
H. influenzae
type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal,
tert
-butyl hydroperoxide (
t
-BuOOH), and
S
-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes.
H. influenzae
NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with
t
-BuOOH as the peroxide substrate. The GSH-mediated protection against
t
-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to
t
-BuOOH toxicity in the Ahp-negative
Escherichia coli
strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative
E. coli
. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.
The
Escherichia coli lipB
Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase
Authors: Jordan Sean W. , Cronan, John E. 
Published: February 2003

ABSTRACT

In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein
N
-lipoyltransferase, in
Escherichia coli
and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that
E. coli lipB
mutants lack this enzyme activity, a finding consistent with
lipB
being the gene that encoded the lipoyltransferase. However, it remained possible that
lipB
encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that
lipB
encodes the lipoyltransferase. A
lipB
temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the
lipB
-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to
lipB
genes in genomic databases was shown to produce a nonfunctional
E. coli
LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.