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SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P 2 position of a peptidomimetic inhibitor. At the P 1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir , and PF-835321 . Our inhibitors contain glutamine isosteres at the P 1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from K i = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC K = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys i , and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P 50 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.145 , and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P 3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

Spontaneous preterm birth (PTB) affects around 11% of births, posing significant risks to neonatal health due to the inflammation at the fetal-maternal interface (FMi). This inflammation disrupts immune tolerance during pregnancy, often leading to PTB. While organ-on-a-chip (OOC) devices effectively mimic the physiology, pathophysiology, and responses of FMi, their relatively low throughput limits their utility in high-throughput testing applications. To overcome this, we developed a three-dimensional (3D)-printed model that fits in a well of a 96-well plate and can be mass-produced while also accurately replicating FMi, enabling efficient screening of drugs targeting FMi inflammation. Our model features two cell culture chambers (maternal and fetal cells) interlinked via an array of microfluidic channels. It was thoroughly validated, ensuring cell viability, metabolic activity, and cell-specific markers. The maternal chamber was exposed to lipopolysaccharides (LPS) to induce an inflammatory state, and proinflammatory cytokines in the culture supernatant were quantified. Furthermore, the efficacy of anti-inflammatory inhibitors in mitigating LPS-induced inflammation was investigated. Results demonstrated that our model supports robust cell growth, maintains viability, and accurately mimics PTB-associated inflammation. This high-throughput 3D-printed model offers a versatile platform for drug screening, promising advancements in drug discovery and PTB prevention.

Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects ( n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing.

During the life cycle of enteric bacterium Escherichia coli , it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli . To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

Tumor susceptibility gene 101 (TSG101) is involved in endosomal maturation and has been implicated in the transcriptional regulation of several steroid hormone receptors, although a detailed characterization of such regulation has yet to be conducted. Here we directly measure binding of TSG101 to one steroid hormone receptor, the glucocorticoid receptor (GR). Using biophysical and cellular assays, we show that the coiled-coil domain of TSG101 (1) binds and folds the disordered N-terminal domain of the GR, (2) upon binding improves the DNA binding of the GR in vitro, and (3) enhances the transcriptional activity of the GR in vivo. Our findings suggest that TSG101 is a bona fide transcriptional co-regulator of the GR and reveal how the underlying thermodynamics affect the function of the GR.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.

A new generation of biomaterials are evolving from being biologically inert toward bioactive surfaces, which can further interact with biological components at the nanoscale. Here, we present directed irradiation synthesis (DIS) as a novel technology to selectively apply plasma ions to bombard any type of biomaterial and tailor the nanofeatures needed for in vitro growth stimulation. In this work, we demonstrate for the first time, the influence of physiochemical cues (e.g., self-organized topography at nanoscale) of medical grade Ti 6 Al 4 V results in control of cell shape, adhesion, and proliferation of human aortic smooth muscle stem cells. The control of surface nanostructures was found to be correlated to ion-beam incidence angle linked to a surface diffusive regime during irradiation synthesis with argon ions at energies below 1 keV and a fluence of 2.5 × 10 17 cm -2 . Cell viability and cytoskeleton morphology were evaluated at 24 h, observing an advance cell attachment state on post-DIS surfaces. These modified surfaces showed 84% of cell biocompatibility and an increase in cytoplasmatic protusions ensuring a higher cell adhesion state. Filopodia density was promoted by a 3-fold change for oblique incidence angle DIS treatment compared to controls (e.g., no patterning) and lamellipodia structures were increased more than a factor of 2, which are indicators of cell attachment stimulation due to DIS modification. In addition, the morphology of the nanofeatures were tailored, with high fidelity control of the main DIS parameters that control diffusive and erosive regimes of self-organization. We have correlated the morphology and the influence in cell behavior, where nanoripple formation is the most active morphology for cell stimulation.

Exposure to petrogenic polycyclic aromatic hydrocarbons (PPAHs) is the major human health hazard associated with the Deepwater Horizon oil spill. Alkylated phenanthrenes are the most abundant PPAHs present in the crude oil and could contaminate the food chain. We describe the metabolism of a C 1 -phenanthrene regioisomer 1-methylphenanthrene (1-MP) and a C 2 -phenanthrene regioisomer 9-ethylphenanthrene (9-EP) in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. Side chain hydroxylation of 1-MP and 9-EP was observed as the major metabolic pathway. The formation of 1-(hydroxymethyl)-phenanthrene was confirmed by reference to an authentic synthetic standard. However, formation of the bioactivated sulfate was not detected. Tetraols were also identified as signature metabolites of 1-MP and 9-EP, indicating that metabolic activation occurred via the diol-epoxide pathway. O-Monosulfonated-catechols were discovered as signature metabolites of the o-quinone pathway of metabolic activation of 1-MP and 9-EP, respectively. The identification of O-monosulfonated-catechols supports the metabolic activation of 1-MP and 9-EP by P450 and AKR isozymes followed by metabolic detoxification of the o-quinone through interception of redox cycling by phase II isozymes. The signature metabolites identified could be used as biomarkers of human exposure to 1-MP and 9-EP resulting from oil spills.

"Science" is under increasing amounts of scrutiny. Concerns about reproducibility, reduced institutional support, and intensified competition has been highlighted in recent years, but segregated science endangers scientific discovery above all. Segregated science can be interdisciplinary (biology vs psychology) or intradisciplinary (behaviorism vs cognitive psychology). The advancement of science and public knowledge depends on the unification of all disciplines to better understand the phenomena scientists study. We suggest that engendering collaborations across scientific disciplines produces better-designed research and appropriate interpretations, and increases career-long success. I am not a cognitive psychologist, behaviorist, or biologist because I am a neuroscientist.

Exposure to petrogenic polycyclic aromatic hydrocarbons (PPAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. C4-Phenanthrenes are representative PPAHs present in the crude oil and could contaminate the seafood. We describe the metabolism of a C4-phenanthrene regioisomer retene (1-methyl-7-isopropyl-phenanthrene) in human HepG2 cells as a model for metabolism in human hepatocytes. Retene because of its sites of alkylation cannot be metabolized to a diol-epoxide. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. O-Monosulfonated-retene-catechols were discovered as signature metabolites of the ortho-quinone pathway of PAH activation catalyzed by aldo-keto reductases. We also found evidence for the formation of bis-ortho-quinones where the two dicarbonyl groups were present on different rings of retene. The identification of O-monosulfonated-retene-catechol and O-bismethyl-O-monoglucuronosyl-retene-bis-catechol supports metabolic activation of retene by P450 and aldo-keto reductase isozymes followed by metabolic detoxification of the ortho-quinone through interception of redox cycling by catechol-O-methyltransferase, uridine 5'-diphospho-glucuronosyltransferase, and sulfotransferase isozymes. We propose that catechol conjugates could be used as biomarkers of human exposure to retene resulting from oil spills.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of l-glutamate and in animals is highly regulated. GDH in hyperinsulinism/hyperammonemia syndrome patients lacks GTP inhibition, resulting in hypersecretion of insulin upon protein consumption. This suggests insulin secretion could be stimulated with GDH activators. A high-throughput screen yielded one potent activator, N1-[4-(2-aminopyrimidin-4-yl)phenyl]-3-(trifluoromethyl)benzene-1-sulfonamide (75-E10). 75-E10 is ∼1000-fold more efficacious than the synthetic activator, BCH, and is at least as effective as ADP. 75-E10 compound is highly effective at alleviating GTP inhibition and may be binding to the ADP site. Unlike ADP, 75-E10 is activated over a broad range of conditions.

Exposure to polycyclic aromatic hydrocarbons (PAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. C1-chrysenes are representative PAHs present in the crude oil and have been detected in contaminated sea food in amounts that exceed their permissible safety thresholds. We describe the metabolism of the most carcinogenic C1-chrysene regioisomer, 5-methylchrysene (5-MC), in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. 5-MC-tetraol, a signature metabolite of the diol-epoxide pathway, was identified as reported previously. Novel O-monosulfonated-5-MC-catechol isomers and O-monomethyl-O-monosulfonated-5-MC-catechol were discovered, and evidence for their precursor ortho-quinones was obtained. The identities of O-monosulfonated-5-MC-1,2-catechol, O-monomethyl-O-monosulfonated-5-MC-1,2-catechol, and 5-MC-1,2-dione were validated by comparison to authentic synthesized standards. Dual metabolic activation of 5-MC involving the formation of bis-electrophiles, i.e., a mono-diol-epoxide and a mono-ortho-quinone within the same structure, bis-diol-epoxides, and bis-ortho-quinones is reported for the first time. Evidence was also obtained for minor metabolic conversion of 5-MC to form monohydroxylated-quinones and bis-phenols. The identification of 5-MC-tetraol, O-monosulfonated-5-MC-1,2-catechol, O-monomethyl-O-monosulfonated-5-MC-1,2-catechol, and 5-MC-1,2-dione supports metabolic activation of 5-MC by P450 and AKR isozymes followed by metabolic detoxification of the ortho-quinone through interception of redox cycling by COMT and SULT isozymes. The major metabolites, O-monosulfonated-catechols and tetraols, could be used as biomarkers of human exposure to 5-MC resulting from oil spills.

Breast tumors often show profound sensitivity to exogenous oxidative stress. Investigational agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) induces aryl hydrocarbon receptor (AhR)-mediated DNA damage in certain breast cancer cells. Since AhR agonists often elevate intracellular oxidative stress, we hypothesize that 5F 203 increases reactive oxygen species (ROS) to induce DNA damage, which thwarts breast cancer cell growth. We found that 5F 203 induced single-strand break formation. 5F 203 enhanced oxidative DNA damage that was specific to breast cancer cells sensitive to its cytotoxic actions, as it did not increase oxidative DNA damage or ROS formation in nontumorigenic MCF-10A breast epithelial cells. In contrast, AhR agonist and procarcinogen benzo[a]pyrene and its metabolite, 1,6-benzo[a]pyrene quinone, induced oxidative DNA damage and ROS formation, respectively, in MCF-10A cells. In sensitive breast cancer cells, 5F 203 activated ROS-responsive kinases: c-Jun-N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38). AhR antagonists (alpha-naphthoflavone, CH223191) or antioxidants (N-acetyl-l-cysteine, EUK-134) attenuated 5F 203-mediated JNK and p38 activation, depending on the cell type. Pharmacological inhibition of AhR, JNK, or p38 attenuated 5F 203-mediated increases in intracellular ROS, apoptosis, and single-strand break formation. 5F 203 induced the expression of cytoglobin, an oxidative stress-responsive gene and a putative tumor suppressor, which was diminished with AhR, JNK, or p38 inhibition. Additionally, 5F 203-mediated increases in ROS production and cytoglobin were suppressed in AHR100 cells (AhR ligand-unresponsive MCF-7 breast cancer cells). Our data demonstrate 5F 203 induces ROS-mediated DNA damage at least in part via AhR, JNK, or p38 activation and modulates the expression of oxidative stress-responsive genes such as cytoglobin to confer its anticancer action.

Thermodynamic and structural characteristics of the Escherichia coli DnaT protein trimerization reaction have been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. Binding of magnesium to the DnaT monomers regulates the intrinsic affinity of the DnaT trimerization reaction. Comparison between the DnaT trimer and the isolated N-terminal core domain suggests that magnesium binds to the N-terminal domain but does not associate with the C-terminal region of the protein. The magnesium binding process is complex and involves approximately three Mg(2+) cations per protein monomer. The observed effect seems to be specific for Mg(2+). In the examined salt concentration range, monovalent cations and anions do not affect the trimer assembly process. However, magnesium affects neither the cooperativity of the trimerization reaction nor the GnHCl-induced trimer dissociation, strongly indicating that Mg(2+) indirectly stabilizes the trimer through the induced changes in the monomer structures. Nevertheless, formation of the trimer also involves specific conformational changes of the monomers, which are independent of the presence of magnesium. Binding of Mg(2+) cations dramatically changes the thermodynamic functions of the DnaT trimerization, transforming the reaction from a temperature-dependent to temperature-independent process. Highly cooperative dissociation of the trimer by GnHCl indicates that both interacting sites of the monomer, located on the N-terminal core domain and formed by the small C-terminal region, are intimately integrated with the entire protein structure. In the intact protein, the C-terminal region most probably interacts with the corresponding binding site on the N-terminal domain of the monomer. Functional implications of these findings are discussed.

The oligomerization reaction of the Escherichia coli DnaT protein has been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. In solution, DnaT exists as a monomer-trimer equilibrium system. At the estimated concentration in the E. coli cell, DnaT forms a mixture of the monomer and trimer states with a 3:1 molar ratio. In spite of the modest affinity, the trimerization is a highly cooperative process, without the detectable presence of the intervening dimer. The DnaT monomer consists of a large N-terminal core domain and a small C-terminal region. The removal of the C-terminal region dramatically affects the oligomerization process. The isolated N-terminal domain forms a dimer instead of the trimer. These results indicate that the DnaT monomer possesses two structurally different, interacting sites. One site is located on the N-terminal domain, and two monomers, in the trimer, are associated through their binding sites located on that domain. The C-terminal region forms the other interacting site. The third monomer is engaged through the C-terminal regions. Surprisingly, the high affinity of the N-terminal domain dimer indicates that the DnaT monomer undergoes a conformational transition upon oligomerization, involving the C-terminal region. These data and the high specificity of the trimerization reaction, i.e., lack of any oligomers higher than a trimer, indicate that each monomer in the trimer is in contact with the two remaining monomers. A model of the global structure of the DnaT trimer based on the thermodynamic and hydrodynamic data is discussed.

Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein--double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer--single-stranded DNA (ssDNA) complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3'-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain--N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg(2+), the association of the cofactors proceeds as a sequential three-step process [Formula: see text] The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)(2), which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)(2), while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

The kinetic mechanism of NTP binding and hydrolysis by the Escherichia coli replicative helicase, the DnaB protein, in the absence and presence of the single-stranded DNA (ssDNA), has been quantitatively examined using the rapid quench-flow technique, under single-turnover conditions. In the case of both the free helicase and the enzyme-ssDNA complexes, the mechanism is independent of the type of base of the cofactor or the DNA; the bimolecular association is followed by the reversible chemical hydrolysis and subsequent conformational transition of the enzyme-product complex. The NTP hydrolysis step is significantly faster for the purine than for the pyrimidine cofactor, both in the absence and in the presence of the DNA. The temperature effect indicates that the nature of intermediates of the purine nucleotide, ATP, is different from the nature of the analogous intermediates of the pyrimidine nucleotide, CTP. Nevertheless, both types of cofactors seem to approach a similar "exit" state at the end of the reaction. The effect of ssDNA on the kinetics of NTP hydrolysis depends on the type of nucleotide cofactor and the base composition of the DNA and is centered at the hydrolysis step. Homoadenosine ssDNA oligomers are particularly effective in increasing the hydrolysis rate. The allosteric signal from the DNA, which activates the NTP hydrolysis, comes predominantly from the strong DNA-binding subsite. The role of the weak DNA-binding subsite is to modulate the allosteric effect of the strong subsite. The significance of these results for the mechanism of the free energy transduction by the DnaB helicase is discussed.

Interactions of nucleotide cofactors with both protein components of the Escherichia coli DnaB helicase complex with the replication factor, the DnaC protein, have been examined using MANT-nucleotide analogues. At saturation, in all examined stationary complexes, including the binary, DnaB-DnaC, and tertiary, DnaB-DnaC-ssDNA, complexes, the helicase binds six cofactor molecules. Thus, protein-protein and protein-DNA interactions do not affect the maximum stoichiometry of the helicase-nucleotide interactions. The single-stranded DNA dramatically increases the ATP analogue affinity, while it has little effect on the affinity of the NDP analogues, indicating that stationary complexes reflect allosteric interactions between the DNA- and NTP-binding site prior to the cofactor hydrolysis step and subsequent to product release. In the binary complex, the DnaC protein diminishes the intrinsic affinity and increases the negative cooperativity in the cofactor binding to the helicase; an opposite effect of the protein on the cofactor-helicase interactions occurs in the tertiary complex. The DnaC protein retains its nucleotide binding capability in the binary and tertiary complexes with the helicase. Surprisingly, the DnaC protein-nucleotide interactions, in the binary and tertiary complexes, are characterized by positive cooperativity. The DnaC assembles on the helicase as a hexamer, which exists in two conformational states and undergoes an allosteric transition, induced by the cofactor. Cooperativity of the allosteric transition depends on the structure of the phosphate group of the nucleotide. The significance of the results for the DnaB-DnaC complex activities is discussed.

Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB-DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB-DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the antagonistic allosteric effect of NTP and NDP on the ssDNA affinity of the enzyme. The protein changes the engagement of the DNA-binding subsites of the helicase in interactions with the nucleic acid, depending on the structure of the phosphate group of the present nucleotide cofactor and profoundly affects the structure of the bound DNA. Moreover, the ssDNA affinity of the helicase in the DnaB-DnaC complex is under the control of the nucleotide-binding site of the DnaC protein. The protein does not affect the NTP hydrolysis mechanism of the helicase. Nevertheless, the rate of the chemical step is diminished in the DnaB-DnaC complex. In the tertiary DnaB-DnaC-ssDNA complex, the ssDNA changes the internal dynamics between intermediates of the pyrimidine cofactor, in a manner independent of the base composition of the DNA, while the hydrolysis step of the purine cofactor is specifically stimulated by the homoadenosine ssDNA. The significance of these results for functional activities of the DnaB-DnaC complex is discussed.

The structure of the complex of the hexameric replicative helicase RepA protein of plasmid RSF1010 with ssDNA has been examined using the fluorescence energy transfer and analytical ultracentrifugation methods. We utilized the fact that the RepA monomer contains a single, natural cysteine residue. The cysteine residue has been modified with a fluorescent marker, which serves as the donor to the acceptor placed in different locations on the DNA. Using the two independent fluorescence donor-acceptor pairs and different DNA oligomers, we provide direct evidence that, in the complex with the enzyme, the ssDNA passes through the inner channel of the RepA hexamer. In the stationary complex, the RepA hexamer assumes a strictly single orientation with respect to the polarity of the sugar-phosphate backbone of the nucleic acid, with the large domain of protomers facing the 3' end of the bound DNA. Interactions with the helicase induce profound changes in the structure of the bound DNA, and these changes are predominantly localized in the proper DNA-binding site. The heterogeneity of the structure of the bound DNA reflects the heterogeneous structure of the total RepA helicase DNA-binding site. This is in excellent agreement with the thermodynamic data. The structure of the RepA hexamer, in solution, differs considerably from the crystal structure of the enzyme. Both fluorescence energy transfer and analytical ultracentrifugation data indicate a significant conformational flexibility of the RepA hexamer. Implications of these results for the mechanism of interactions of the hexameric helicase with the DNA are discussed.

Energetics and specificity of interactions between the African swine fever virus polymerase X and gapped DNA substrates have been studied, using the quantitative fluorescence titration technique. Stoichiometries of pol X complexes, with the DNA substrates, are higher than suggested by NMR studies. This can be understood in the context of the functionally heterogeneous organization of the total DNA-binding site of pol X, which is composed of two DNA-binding subsites. The enzyme forms two different complexes with the gapped DNAs, differing dramatically in affinities. In the high-affinity complex, pol X engages the total DNA-binding site, forming the gap complex, while in the low-affinity the enzyme binds to the dsDNA parts of the gapped DNA, using only one of the DNA-binding subsites. As a result, the net number of ions released in the gap complex formation is significantly larger than in the binding of the dsDNA part. In the presence of Mg+2, pol X shows a strong preference for the ssDNA gaps having one and two nucleotides. Recognition of the short gaps already occurs in the ground state of the enzyme-DNA complex. Surprisingly, the specific structure necessary to recognize the short gaps is induced by magnesium binding to the enzyme. In the absence of Mg+2, pol X looses its selectivity for short ssDNA gaps. Pol X binds gapped DNAs with considerable cooperative interactions, which increase with the decreasing gap size. The functional implications of these findings for ASFV pol X activities are discussed.

Allosteric interactions between the strong and weak nucleotide-binding sites and the total and proper single-stranded (ss)DNA-binding sites of the Escherichia coli PriA helicase have been analyzed using the fluorescence titration technique. Binding of the DNA exclusively to the proper DNA-binding site of the helicase, profoundly affects the intrinsic affinities of both nucleotide-binding sites, indicating a direct communication between the nucleotide-binding sites and the proper DNA-binding site. The communication involves conformational changes of the entire protein molecule. Nevertheless, the bound DNA differently affects the structures of the strong and weak nucleotide-binding sites. While the polarity of the strong site is moderately diminished, the polarity of the weak site is dramatically increased, indicating an intimate involvement of the weak site in controlling the helicase interactions with the DNA. The strong site does not directly control the DNA affinity of the enzyme. Only when the helicase has both nucleotide-binding sites saturated with ADP but not with ATP analogues does the enzyme have an increased affinity for the ssDNA, indicating that the control of ssDNA affinity involves a coordinated action of both nucleotide-binding sites and depends upon the phosphate group of the bound cofactor. A dramatic increase of the DNA affinity, when the DNA encompasses the total DNA-binding site of the enzyme, with both nucleotide-binding sites saturated with ADP or NDP, indicates that an additional area of the protein within the total DNA-binding site becomes engaged in interactions with the DNA. The significance of these results for the enzyme activities in the DNA unwinding and recognition is discussed.

Interactions of the Escherichia coli PriA helicase with nucleotide cofactors have been studied using the fluorescence titration and analytical ultracentrifugation techniques. Binding of unmodified cofactors was characterized by the fluorescence competition titration method. The obtained data establish that at saturation the PriA helicase binds two nucleotide molecules per protein monomer. This result corroborates with the primary structure of the protein, which contains sequence motifs implicated as putative nucleotide-binding sites. The intrinsic affinities of the binding sites differ by 2-4 orders of magnitude. Thus, the PriA helicase has a strong and a weak nucleotide-binding site. The binding sites differ dramatically in their properties. The strong site is highly specific for adenosine cofactors, while the weak site shows very modest base specificity. The affinities of the strong and weak binding sites for ATP are lower than the affinities for ADP, although both sites have similar affinity for the inorganic phosphate group. Unlike the weak site, the affinity of the strong site profoundly depends on the structure of the phosphate group of the ATP cofactor. Binding of unmodified nucleotides indicates the presence of positive cooperative interactions between bound cofactors (i.e., the existence of communication between the two sites). Magnesium cations are specifically involved in controlling the cofactor affinity for the strong site, while the affinity of the weak site is predominantly determined by interactions between the phosphate group and ribose regions of the cofactor and the protein matrix. The significance of these results for the activities of the PriA helicase is discussed.

Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.

The interactions of nucleotides with RepA hexameric helicase from plasmid RSF1010 have been examined using nucleotide analogues, TNP-ADP, TNP-ATP, and MANT-ADP. The binding of the analogues is accompanied by strong quenching of the protein fluorescence. A quantitative fluorescence titration method has been applied to analyze the interactions, independent of any assumptions of proportionality between the fluorescence quenching and the average degree of binding. The fluorescence quenching as a function of the average degree of binding is expressed by an empirical function that enables analysis of the data, without the necessity of determining quenching parameters for different complexes. At saturation, the RepA hexamer binds six nucleotide molecules, indicating that each subunit of the hexamer can engage in interactions with the cofactor. The nucleotide macroscopic affinity decreases with the increasing degree of binding, indicating heterogeneity among the binding sites. A statistical thermodynamic hexagon model provides an excellent description of the binding process and requires only two interaction parameters, the intrinsic binding constant, K, and cooperativity parameter, sigma. The heterogeneity in affinity reflects negative cooperative interactions between the binding sites. Analyses of the data provide clear evidence that the alternative model of two independent classes of binding sites does not describe the nucleotide binding. Such a model cannot account for both, the binding isotherms and the dependence of the fluorescence quenching upon the degree of binding. Thus, cooperative interactions between the nucleotide-binding sites are an intrinsic property of the RepA helicase. The presence of the cooperative interactions indicates significant communication among the subunits of the helicase.

Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of approximately 10 and approximately 1000 higher than the value observed for ADP and PO(4)(-). The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the gamma phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg(2+), to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg(2+) cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg(2+) has a dual function in nucleotide-helicase interactions. At low [Mg(2+)], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg(2+)], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base.

Kinetics of cooperative binding of rat polymerase beta to a double-stranded DNA has been studied using the fluorescence stopped-flow techniques. The data have been analyzed by an approach developed to examine complete kinetics of cooperative large ligand binding to a one-dimensional lattice. The method is based on using the smallest possible system that preserves key ingredients of cooperative binding; i.e., at saturation, the lattice can accept only two ligand molecules. It allows the identification of collective amplitudes as well as amplitudes describing particular normal modes of the reaction. The mechanism of the intrinsic binding of pol beta to the dsDNA is different from the analogous mechanism for the ssDNA. The difference originates from different enzyme orientations in the corresponding complexes. Intrinsic binding to the dsDNA includes only two sequential steps: a very fast bimolecular association followed by an energetically favorable conformational transition of the complex. The transition following the bimolecular step does not facilitate the engagement of the enzyme in cooperative interactions. Its role seems to be reinforcing the affinity of the bimolecular step. Salt and magnesium cations affect both the bimolecular step and the conformational transition. As a result, the bimolecular step is less sensitive to the increased salt concentration, allowing the enzyme to preserve its initial dsDNA affinity. The changing character of cooperative interactions between bound enzyme molecules as a function of NaCl concentration and MgCl(2) concentration does not affect the binding mechanism. The engagement in cooperative interactions is approximately 3-4 orders of magnitude slower than the conformational transition of the DNA-bound polymerase. The importance of the obtained results for the pol beta activities is discussed.

Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor. The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone. We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity. To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein in Escherichia coli. Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein. Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein. The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of (125)I-CGRP and (125)I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits (125)I-CGRP and (125)I-AM binding to rat uterus in a dose-dependent fashion (IC(50) = 0.25 and 0.29 muM, respectively). Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.

Kinetics of the Escherichia coli PriA helicase interactions with the ssDNA has been studied, using the fluorescence stopped-flow technique. Experiments have been performed with a series of fluorescent etheno derivatives of ssDNA adenosine oligomers, differing in the number of nucleotide residues. The PriA helicase binds the ssDNA in the sequential process defined by [reaction: see text]. In the first step, the enzyme associates fast with the ssDNA without inducing conformational changes in the DNA. The dependence of the partial equilibrium constant, characterizing the first step, upon the length of the ssDNA strictly reflects the statistical relationship between the size of the DNA-binding site and the number of potential binding sites on the ssDNA. Only the DNA-binding site that encompasses 6.3 +/- 1 residues is directly involved in interactions. The site is located on a structural domain allowing the enzyme to efficiently search and recognize small patches of the ssDNA. Intramolecular steps are independent of the ssDNA length and accompanied by changes in the DNA structure. Salt and glycerol effects on the studied kinetics indicate a very different nature of the intermediates. While the bimolecular step is characterized by net ion release and water uptake, net ion uptake and water release accompany intramolecular transitions. Specific ion binding stabilizes the helicase-ssDNA complex in (P)(2) and (P)(3) intermediates. However, magnesium and AMP-PNP do not affect the mechanism of enzyme-ssDNA interactions. The sequential character of the mechanism indicates that the enzyme does not exist in a preequilibrium conformational transition prior to the DNA binding.

Global conformational and oligomeric states of the Escherichia coli replicative factor DnaC protein in the absence and presence of magnesium and nucleotide cofactors, ATP and ADP, and their fluorescent analogues, MANT-ATP and MANT-ADP, have been examined using analytical sedimentation velocity and time-dependent fluorescence anisotropy techniques. In solution, the DnaC protein exists exclusively as a monomer over a large protein concentration range. The value of s(degrees) (20, w)= 2.45 +/- 0.07 S indicates that the protein molecule has an elongated shape. When modeled as a prolate ellipsoid of revolution, the hydrated DnaC protein has an axial ratio of 4.0 +/- 0.6 with long axis a = 112 A and the short axis b = 28 A, respectively. The presence of magnesium or nucleotide cofactors, ATP or ADP, does not affect the global conformation of the protein and its monomeric state. These data indicate that recently found cooperative interactions between the DnaC molecules, in the complex with the DnaB helicase, are induced by the binding to the helicase, i.e., they are not the intrinsic property of the DnaC protein. Fluorescence anisotropy decays of the DnaC-MANT-ATP and DnaC-MANT-ADP complexes indicate that the protein has a rigid global structure on the nanosecond time scale, little affected by the nucleotide cofactors. Nevertheless, the complex with ATP has a more flexible structure, while the complex with ADP is more rigid, with the protein molecule assuming a more elongated shape. Magnesium exerts control only on the complex with the ATP analogue. In the absence of magnesium, the ATP analogue is firmly held in the binding site. In the presence of Mg(2+), this fixed location is released and the analogue is allowed to assume a flexible conformational state. The significance of the results for the functioning of the DnaC protein is discussed.

A quantitative characterization of the structure and energy of the denatured states of proteins represents the cornerstone to a molecular-level understanding of both protein stability and fold specificity. Recent studies have revealed a significant bias in unstructured peptides toward the polyproline II (P(II)) conformation, even when no prolines are present in the sequence. This indicates that the P(II) conformation is a dominant component of the denatured states of proteins, although a quantitative description of the component enthalpy and entropy functions associated with this conformation (i.e., the thermodynamic mechanism) has thus far proven elusive. An experimental system has been designed that, when analyzed with high-precision isothermal titration calorimetry, provides direct access to the residue-specific thermodynamics of the P(II) structure formation in disordered proteins and peptides. Here, it is shown that the P(II) bias is driven by a favorable and significant enthalpy (Deltah) of -1.7 kcal mol(-1) residue(-1), which is partially offset by an unfavorable entropy (TDeltas) of -0.7 kcal mol(-1) residue(-1), relative to the ensemble of disordered conformations of the molecule. In addition to impacting dramatically the interpretation of thermal denaturation experiments, these experimental values form the framework of a quantitative energetic description of the denatured states of proteins.

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844-3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857-11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 microM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide approximately cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the DeltaG for CRP-DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.

The mosquito-borne West Nile (WNV) and dengue 2 (DEN2V) viruses and tick-borne Langat (LGTV) and Omsk hemorrhagic fever (OHFV) viruses are arthropod-borne flaviviruses (family Flaviviridae, genus Flavivirus). These viruses are quite similar at both the nucleotide and amino acid level, yet they are very divergent in their biological properties and in the diseases they cause. The objective of this study was to examine the putative receptor-binding domains of the flaviviruses, the envelope (E) protein domain III (D3), which assume very similar structures either as part of the whole envelope protein or as individual entities, and to define the biophysical properties that distinguish among these viruses. Circular dichroism and Fourier transform infrared spectroscopy were employed to monitor the solution structure of these proteins. While the spectroscopic results found that the D3 from each of these viruses is composed of either beta-sheets or beta-turns, which is consistent with X-ray crystal data for tick-borne encephalitis and dengue viruses, these results reveal that recombinant D3s (rED3s) derived from tick-borne flaviviruses (LGT-rED3 and OHF-rED3) were similar to each other, while those from mosquito-borne flaviviruses (WN-rED3 and DEN-rED3) were similar to each other yet distinct from rED3 of the tick-borne viruses. Protein dynamic studies probed by fluorescence quenching and hydrogen/deuterium exchange found that the rED3s are dynamic entities. The tick-borne proteins again exhibit very similar dynamic properties, which are different from the mosquito-borne proteins. The WN-rED3 is significantly less stable than the other three rED3s. Overall, these differences in biophysical properties correlate with biological properties of these viruses that tick-borne flaviviruses are more stable than mosquito-borne flaviviruses.

The cAMP receptor protein (CRP) of Escherichia coli is a transcription factor. The affinity of CRP for a specific DNA sequence is significantly enhanced as a consequence of the binding of the allosteric effector, cAMP. The hinge region, particularly residues 136 and 138, connecting the cAMP and DNA binding domains of CRP has been proposed to play essential roles in transmitting the allosteric signals. To probe the specific role of residue 138, eight D138 mutants and wild-type CRP were tested for their ability to bind the lac26 and gallac26 promoter sequences in this study. A correlation was established between DNA binding affinity and side chain solvation free energy, namely, an increasing specific DNA affinity with an increasing hydrophilicity of the side chain of residue 138. In addition, a linear correlation was found between DNA binding affinity and the energetics of subunit assembly. The ability of CRP to distinguish between cAMP and cGMP as an allosteric activator of DNA binding is weakened with higher energetics of subunit assembly. This correlation indicates that quaternary constraint leads to a constraint of the DNA binding domain. This observation is consistent with the concept that an optimum quaternary structural constraint is important in CRP exhibiting its allosteric properties. The stability of CRP was monitored by Trp fluorescence and circular dichroism in the presence of guanidine hydrochloride. These spectroscopic data revealed nonidentical denaturation profiles. Since the Trp residues are located exclusively in the beta-roll cyclic nucleotide binding domain, the denaturation profiles reveal the stability of the beta-roll structure. This study produces another linear correlation between DNA binding affinity in the presence of cAMP and cGMP and the stability of the beta-roll; namely, the stability of the beta-roll structure leads to a decrease in DNA binding affinity. All these correlations indicate the importance of structural stability and dynamics in the ability of CRP to manifest its intrinsic allosteric properties.

The tertiary structure of template-primer and gapped DNA substrates in the complex with rat polymerase beta (pol beta) has been examined using the fluorescence energy transfer method based on the multiple donor-acceptor approach. In these studies, we used DNA substrates labeled at the 5' end of the template strand and the 5' end of the primer with the fluorescent donor and/or acceptor. Measurements of the enzyme complex with the template-primer DNA substrate having a ten nucleotide long ssDNA extension indicate that the distance between the 5' end of the template strand and the 5' end of the primer decreases by approximately 9.8 A as compared to the free nucleic acid. Analogous experiments with the template-primer substrate, having the ssDNA extension with five nucleotide residues, show approximately 6.6 A distance decrease. Such large distance decreases indicate that the DNA is significantly bent in the binding site. Analysis of the data indicates that the bending occurs between the third and the fourth nucleotide of the ssDNA extension. The entire template strand is at the bend angle Theta(TP) = 85 +/- 7 degrees with respect to the dsDNA part of the DNA molecule. In the polymerase complex with the gapped DNA, the distance between the 5' ends of the DNA and the bend angle are 66 +/- 2.2 A and 65 +/- 6 degrees, respectively. These values are very similar to the same distance and bend angle of the gap complex in the crystal structure of the co-complex. The presence of the 5'-terminal PO(4)(-) group downstream from the primer does not affect the tertiary conformation of the gapped DNA, indicating that the effect of the phosphate group is localized at the ssDNA gap.

Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed (15)N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed.

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.

The activated Escherichia coli cAMP receptor protein, CRP, is capable of regulating the expression of more than 20 genes by binding to specific DNA sites. DNA bending is an important structural feature that has been observed in the regulatory mechanism of gene expression by CRP. On the basis of the results of the fluorescence energy transfer study of the gal P1 promoter, gal bends asymmetrically upon binding to CRP, although DNA bends symmetrically in the CRP-lac complex. The flanking sequence proximal to the TGTGA motif is involved in a sharper bend than the other side with an overall bending angle of approximately 90-125 degrees, without wrapping around the CRP molecule. To understand the factors that control the symmetry in DNA bending, a series of DNA sequences was tested to dissect the contribution of half-sites and flanking sequences, using the natural gal P1 and lac P1 sequences as initial targets. The extent of DNA bending induced by CRP was monitored by the difference in fluorescence anisotropy between free DNA and the DNA-CRP complex. The extent of bending was sequence-dependent, and most importantly, the symmetry of bending was a function of the symmetry of the DNA sequence. For example, in the lac promoter the two binding half-sites (TGTGA and TCACT) were almost symmetric as an inverted repeat. The recognition F-helices of the two CRP subunits would bind to these half-sites with a 2-fold symmetry. The flanking sequences (ATAAA and CATTA) were almost identical mirror images. Thus, they are expected to bend in a similar manner. Finally, the sequence symmetry properties of a series of natural CRP promoters were analyzed. A strong tendency for symmetry sequence was encoded in class I promoter sites but not in class II promoter sites. Results from this analysis support the conclusion that the geometry of the CRP-DNA complex plays a major role in determining the molecular mechanism in gene transcription.

E. coli cyclic AMP receptor protein, CRP, is a modular protein that consists of a covalent linkage of two common structural domains. To probe the mechanism for intramolecular communications and to define the unique properties acquired by covalent linkage, the structural, and functional properties of the cAMP- and DNA-binding domains of CRP were studied separately as two independent polypeptides. The N-terminal cAMP-binding domain (alpha-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by beta-sheets. The C-terminal DNA-binding domain, designated as beta-CRP, consists of mostly alpha-helices. The residues of S-CRP and CH-CRP are from 1 to 116 and 1 to 136 of intact wild-type CRP, and those of beta-CRP are from 108 to 209. The secondary structures of alpha-CRP and beta-CRP were monitored by FT-IR, and they are similar to those of the corresponding parts in intact wild-type CRP. Results from hydrogen-deuterium exchange experiments indicated that beta-CRP is more dynamic than alpha-CRP. In an earlier study, it was shown that alpha-CRP retains the function of binding cAMP [Heyduk, E., et al. (1992) Biochemistry 31, 3682-3688]. beta-CRP was able to bind to DNA, although only weakly, and was not sequence specific. Thus, a covalent linkage between the two domains is essential for the realization of the intramolecular signal transmission between the domains triggered by ligand binding. The acquisition of this unique property is intimately associated with the dynamics of the molecule.

The transcription factor cyclic AMP receptor protein, CRP, regulates the operons that encode proteins involved in translocation and metabolism of carbohydrates in Escherichia coli. The structure of the CRP-cAMP complex reveals the presence of two sets of cAMP binding sites. Solution biophysical studies show that there are two high-affinity and two low-affinity binding sites, to which the binding of cAMP is characterized by varying degrees of cooperativity. A stoichiometry of four implies that potentially CRP can exist in five conformers with different numbers of bound cAMP. These conformers may exhibit differential affinities for specific DNA sequences. In this study, the affinity between DNA and each conformer of D53H CRP was defined through a dissection of the thermodynamic linkage scheme that included all the conformers. Loading of the high- and low-affinity sites with cAMP leads to high and low affinity for DNA, respectively. The specific magnitude of the binding constants of these conformers is DNA sequence dependent. The various association constants defined by the present study provide a solution to address an enigma of the CRP system, namely, the 3 orders of magnitude difference between the cAMP binding constants determined by in vitro studies and the cAMP concentration regime to which the bacteria respond. Under physiological conditions, the apo-CRP-DNA complex is the dominant species. As a consequence of the 1000-fold stronger affinity of cAMP to the apo-CRP-DNA complex than that to CRP, the relevant reaction is the binding of cAMP to this DNA-protein complex. The binding constant is of the order of 10(7) M(-)(1), the same concentration regime as that of cellular concentration of cAMP. In addition, under physiological conditions the species that binds to the lac and gal operons is predicted to be CRP-(cAMP)(1). A comparison of parameters between the wild type and the mutant CRP shows that the mutation apparently shifts the various thermodynamically linked equilibria without a change in the basic mechanism that governs CRP activities. Thus, the conclusions derived from a study of the mutant are relevant to wild-type CRP. A dissection of the individual binding constants in this multiequilibria reaction scheme leads to a definition of the mechanism of action of this transcription factor.

The binding of adenosine 3',5'-cyclic monophosphate (cAMP) and its nonfunctional analogue, guanosine 3',5'-cyclic monophosphate (cGMP), to the adenosine 3',5'-cyclic monophosphate receptor protein (CRP) from Escherichia coli was investigated by means of fluorescence and isothermal titration calorimetry (ITC) at pH 7.8 and 25 degrees C. A biphasic fluorescence titration curve was observed, confirming the previous observation reported by this laboratory (Heyduk and Lee (1989) Biochemistry 28, 6914-6924). However, the triphasic titration curve obtained from the ITC study suggests that the cAMP binding to CRP is more complicated than the previous conclusion that CRP binds sequentially two molecules of cAMP with negative cooperativity. The binding data can best be represented by a model for two identical interactive high-affinity sites and one low-affinity binding site. Unlike cAMP, the binding of cGMP to CRP exhibits no cooperativity between the high-affinity sites. The effects of mutations on the bindings of cAMP and cGMP to CRP were also investigated. The eight CRP mutants studied were K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L. These sites are located neither in the ligand binding site nor at the subunit interface. The binding was monitored by fluorescence. Although these mutations are at a variety of locations in CRP, the basic mechanism of CRP binding to cyclic nucleotides has not been affected. Two cyclic nucleotide molecules bind to the high-affinity sites of CRP. The cooperativity of cAMP binding is affected by mutation. It ranges from negative to positive cooperativity. The binding of cGMP shows none. With the exception of the T127L mutant, the free energy change for DNA-CRP complex formation increases linearly with increasing free energy change associated with the cooperativity of cAMP binding. This linear relationship implies that the protein molecule modulates the signal in the binding of cAMP, even though the mutation is not directly involved in cAMP or DNA binding. In addition, the significant differences in the amplitude of fluorescent signal indicate that the mutations also affect the surface characteristics of CRP. These results imply that these mutations are not perturbing specific pathways of signal transmission. Instead, these results are more consistent with the concept that CRP exists as an ensemble of native states, the distribution of which can be altered by these mutations.

The kinetic mechanism of binding of ATP and ADP fluorescent analogues to the E. coli replicative factor DnaC protein has been studied using the fluorescence stopped-flow technique. The experiments have been performed under pseudo-first-order conditions with respect to the nucleotide cofactor or the DnaC concentration. Three relaxation processes are observed at a large excess of the nucleotide, while only two relaxation processes are detected in the excess of the protein. Such behavior of the kinetic system is a diagnostic indication of the presence of the protein conformational equilibrium prior to the ligand binding. The obtained data indicate that the minimum mechanism that describes the observed kinetics includes the conformational transition of the DnaC protein, prior to nucleotide binding, followed by the two-step, sequential association of the cofactor to only one of the protein conformations, as defined by In the examined solution conditions, the conformation of the DnaC protein is shifted toward the state (DnaC)(2) that binds the nucleotide. The lack of any cofactor binding to the (DnaC)(1) state points to the existence of a stringent locking mechanism of the nucleotide binding-site in the protein. Binding of ATP and ADP analogues obeys the same mechanism, with similar rate constants, indicating that ATP and ADP analogues bind to the same protein conformation. The (C)(1) intermediate dominates the distribution of the DnaC protein population in the presence of cofactors. The formation of (C)(1) is accompanied by a low nucleotide fluorescence increase, indicating a hydrophilic environment around the ribose of bound cofactors. Transition to (C)(2) places the ribose region in a highly hydrophobic environment with relative molar fluorescence intensity approximately 8-fold higher than that of the free cofactor. The significance of these results for the functioning of the DnaC protein is discussed.

The nature of the intermediates in the binding of MANT-ATP and MANT-ADP to the E. coli replicative factor DnaC protein (accompanying paper) has been examined using the fluorescence intensity, anisotropy, and transient dynamic quenching stopped-flow techniques. Using molar fluorescence intensities of individual intermediates of the reaction, we derived the Stern-Volmer equation that provides a direct method to quantitatively address the quenching of the fluorescence of a transient intermediate by an external, neutral quencher. The data indicate that in the first intermediate, (C)(1), the solvent has full access to the MANT group. Thus, the nucleotide-binding site is located on the surface of the protein, fully open to the solvent. Moreover, formation of the first intermediate does not affect the structure of the binding site. On the other hand, in the second intermediate, (C)(2), the entire binding site changes its conformation, resulting in diminished access of the solvent to the bound nucleotide. The time course of the fluorescence anisotropy in the reaction provides direct, unique insight into the mobility of bound nucleotides in each intermediate. The analysis is facilitated by the fact that the anisotropy can be expressed as a function of the relative molar intensities and steady-state anisotropies of the individual intermediates. The major decrease of the nucleotide mobility occurs in the formation of the first intermediate and reflects the fact that the MANT group is immobilized to a similar extent as the ribose region of the bound nucleotides. Transition to the second intermediate and closing of the binding site leads to only a moderate, additional decrease of nucleotide mobility. The temperature effect on the studied interactions indicates that the formation of individual intermediates is accompanied by very different enthalpy and entropy changes predominantly generated from the structural changes of the protein. Analysis of the salt effect indicates that the net release of a single ion, observed in equilibrium studies, occurs in the formation of the first intermediate. The lack of any salt effect on the (C)(1) <--> (C)(2) transition indicates that the closing of the binding site does not include a net ion release or uptake. Moreover, prior to the nucleotide binding, the conformational transition of the DnaC protein is exclusively controlled by the nucleotide binding and release.

The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.

Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the gamma-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-gamma-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are approximately 2 and approximately 4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADP are approximately 2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.

The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues bearing the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, epsilonAMP-PNP and epsilonADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four-steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: [equation in text]. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)(1). Moreover, the value of the bimolecular rate constant, k(1), is 3-4 orders of magnitude lower than the value expected for the diffusion-controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.