Fluorescence Anisotropy Titration Research Articles

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 μM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3.

Human galectin-3 (hGal-3) is involved in a variety of biological processes and is implicated in wide range of diseases. As a result, targeting hGal-3 for clinical applications has become an intense area of research. As a step towards the development of novel hGal-3 inhibitors, we describe a study of the binding of two Se-containing hGal-3 inhibitors, specifically that of di(β-D-galactopyranosyl)selenide (SeDG), in which two galactose rings are linked by one Se atom and a di(β-D-galactopyranosyl)diselenide (DSeDG) analogue with a diseleno bond between the two sugar units. The binding affinities of these derivatives to hGal-3 were determined by 15N-1H HSQC NMR spectroscopy and fluorescence anisotropy titrations in solution, indicating a slight decrease in the strength of interaction for SeDG compared to thiodigalactoside (TDG), a well-known inhibitor of hGal-3, while DSeDG displayed a much weaker interaction strength. NMR and FA measurements showed that both seleno derivatives bind to the canonical S face site of hGal-3 and stack against the conserved W181 residue also confirmed by X-ray crystallography, revealing canonical properties of the interaction. The interaction with DSeDG revealed two distinct binding modes in the crystal structure which are in fast exchange on the NMR time scale in solution, explaining a weaker interaction with hGal-3 than SeDG. Using molecular dynamics simulations, we have found that energetic contributions to the binding enthalpies mainly differ in the electrostatic interactions and in polar solvation terms and are responsible for weaker binding of DSeDG compared to SeDG. Selenium-containing carbohydrate inhibitors of hGal-3 showing canonical binding modes offer the potential of becoming novel hydrolytically stable scaffolds for a new class of hGal-3 inhibitors.

Role of Lys Residue at Position 87 of DREAM in Allosteric Regulation of DREAM's Interactions with KV Channel

Downstream regulatory element antagonist modulator (DREAM) is a 29kDa protein, which interacts with diverse intracellular partners and is involved in many biological processes, namely, pain sensation, gene apoptosis, and modulation of Kv4 voltage channels. Previous research in our group and elsewhere demonstrated that DREAM interacts with the helix-9 of presenilin-1 (PS1HL9), the residue 2-22 (site-1) peptide of Kv4.3, and the residue 70-90 (site-2) peptide of Kv4.3 in a calcium-dependent manner. Molecular dynamics data suggests that Lys at the position 87 forms a salt bridge with Asp 165 and directly involved in the propagation of calcium-triggered structural changes between the C- terminal and N- terminal domain. To determine the impact of Lys 87 on the interdomain communication as well as to characterize its contribution to DREAM stability, Lys 87 was mutated to Ala, and the effects of the mutation on DREAM's interaction with “PS1HL9”, “site-1” peptide, and “site-2” were determined. The results show that Trp residue in DREAM(K87A) is more solvent exposed compared with wild-type suggesting that the absence of the salt bridge destabilizes the protein structure. The emission maximum of 1,8-ANS in the presence of DREAM(K87A) is about 10 nm redshifted indicating that the mutation influences the hydrophobic cavity between the N- and C-terminal domain. The lifetime of Trp and 1,8-ANS are significantly altered in DREAM(K87A) compared with wild-type form, which in agreement with the steady-state data. Fluorescence anisotropy titration data suggest that mutation of Lys to Ala at position 87 of DREAM completely inhibit DREAM's interaction with site-2. We do plan to investigate DREAM(K87A)'s interactions with site-1 and PS1HL9. Data presented here provide insight into the role of Lys residue at position 87 of DREAM regulating DREAM's interaction with intracellular partners.

Truncation, modification, and optimization of MIG6segment 2 peptide to target lung cancer-related EGFR

Human epidermal growth factor receptor (EGFR) plays a central role in the pathological progression and metastasis of lung cancer; the development and clinical application of therapeutic agents that target the receptor provide important insights for new lung cancer therapies. The tumor-suppressor protein MIG6 is a negative regulator of EGFR, which can bind at the activation interface of asymmetric dimer of EGFR kinase domains to disrupt dimerization and then inactivate the kinase (Zhang X. et al. Nature 2007, 450: 741–744). The protein adopts two separated segments, i.e. MIG6segment 1 and MIG6segment 2, to directly interact with EGFR. Here, computational modeling and analysis of the intermolecular interaction between EGFR kinase domain and MIG6segment 2 peptide revealed that the peptide is folded into a two-stranded β-sheet composed of β-strand 1 and β-strand 2; only the β-strand 2 can directly interact with EGFR activation loop, while leaving β-strand 1 apart from the kinase. A C-terminal island within the β-strand 2 is primarily responsible for peptide binding, which was truncated from the MIG6segment 2 and exhibited weak affinity to EGFR kinase domain. Structural and energetic analysis suggested that phosphorylation at residues Tyr394 and Tyr395 of truncated peptide can considerably improve EGFR affinity, and mutation of other residues can further optimize the peptide binding capability. Subsequently, three derivative versions of the truncated peptide, including phosphorylated and dephosphorylated peptides as well as a double-point mutant were synthesized and purified, and their affinities to the recombinant protein of human EGFR kinase domain were determined by fluorescence anisotropy titration. As expected theoretically, the dephosphorylated peptide has no observable binding to the kinase, and phosphorylation and mutation can confer low and moderate affinities to the peptide, respectively, suggesting a good consistence between the computational analysis and experimental assay.

Multiphasic Profiles: Discontinuous Transitions in Conductance-Voltage Data for Ion Channels

Multiphasic profiles, a series of straight lines separated by discontinuous transitions, were first found (Nissen, Annu. Rev. Plant Physiol. 1974) for the concentration-dependence, plotted in linear transformations of the Michaelis-Menten equation, for ion uptake in plants. Reanalyses of recently published data show that conductance-voltage data for ion channels are also well represented as multiphasic. In contrast, the Boltzmann function and other functions giving curvilinear profiles must often be rejected for statistical reasons (very low probabilities by the Runs test that the uneven distribution of points around the profiles is due to chance). Plots of deviates show that the fits to multiphasic profiles are usually better than the fits to curvilinear profiles, i.e. that the data are more precise than apparent from the published plots. As shown by simulation, the better fits to multiphasic profiles are not due to errors (noise) in the data. Adjacent lines are often parallel. The transitions are then necessarily in the form of noncontiguities (jumps). Non-parallel lines are also frequently separated by jumps. Most often, replicate determinations give different multiphasic patterns (number of phases, voltages at which the transitions occur). The use of the averages will then give a meaningless pattern.Whenever the data are sufficiently detailed and precise, multiphasic profiles are also found for a variety of other processes and systems, biological as well as nonbiological: 1) pH profiles, including profiles for non-enzymatic model systems. 2) Binding as determined by fluorescence anisotropy titration or isothermal titration calorimetry (ITC). 3) Folding and unfolding of proteins.Implications for the opening and closing of single biological channels will be discussed.

Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1)

Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca2+)4–CaM inhibits it. CaM-binding RyR1 residues 1975–1999 and 3614–3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR11975–1999 or hRyR13614-3643. Both CaM and its C-domain associated in a calcium-independent manner with hRyR13614–3643 while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR111975–1999 was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR13614–3643 at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR11975–1999 alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation.

The Acidic C-terminal Tail of the GyrA Subunit Moderates the DNA Supercoiling Activity of Bacillus subtilis Gyrase

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.

Unraveling the Mechanism of the Primosome Assembly Process

E. Coli primosome is a multiprotein-DNA complex present at the crossroads of DNA replication and repair where it is used to restart stalled replication fork at the damage sites. The assembly and function of the primosome is controlled by synchronized action of seven proteins, PriA, PriB, DnaT, PriC, DnaB, DnaC, DnaG, and nucleic acid. The presence of two helicases: PriA and DnaB, ensures bidirectional mechanical translocation and/or unwinding while DnaG primase synthesizes oligoribonucleotide primers used by DNA polymerase III holoenzyme.In spite of intensive studies on primosome structure and function over the last two decades, many basic aspects of the primosome assembly process remain ambiguous. Fundamental characteristics, such as the stoichiometry and interactions within the primosome are not well understood. As the result, the molecular mechanism of the assembly process of the primosome is still lacking.Here, using fluorescence intensity, fluorescence anisotropy titration, fluorescence resonance energy transfer and analytical ultracentrifugation methods, we present direct quantitative analysis of the initial steps in primosome assembly, involving PriA, PriB and DnaT proteins and the minimal primosome assembly site (PAS). The obtained results provide a quantitative framework for initiation of primosome formation and elucidation of further steps in the assembly process.

Helicase-Initiated Assembly of the Primosome

A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage ϕX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the PAS structure without detectable cooperative interactions. Binding of the PriB dimer to the PriA - PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Interactions are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA - PriB complex, formed independently of the DNA, is able to directly recognize the PAS structure. Thus, the high-affinity state of PriA for PAS is generated through PriA - PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for initiation of primosome formation and elucidation of further steps in the assembly process.

Analysis of Oligomer Assembly for the GluA2 Amino Terminal Domain

For GluA2, an ionotropic glutamate receptor that mediates excitatory synaptic transmission at CNS synapses, the reported monomer-dimer Kd of the amino-terminal domain (ATD) varies widely, ranging from 1.8 nM to 4 μM.1-3 To investigate causes of discrepancies in the literature, several protein constructs, diverse in glycosylation and overall size, were created and the dimer Kd analyzed by analytical ultracentrifugation with absorbance and interference optics (AUC). Sedimentation velocity (SV) Kds varied from 2 to 11 nM, with size and glycosylation having little effect. At the highest protein concentration examined (33 μM) we did not detect formation of tetramers or larger oligomer species. Steady state fluorescence anisotropy titrations for DyLight405 labelled protein yielded similar results. However, sedimentation equilibrium analysis yielded dissociation constants of 160 to 280 nM. Temperature and pressure variations did not explain this difference. Small amounts of degradation over the several day sedimentation equilibrium protocol was revealed by silver-stained SDS-PAGE and likely underlies the different Kds for sedimentation velocity and sedimentation equilibrium experiments. Our results confirm that the GluA2 ATD forms nM affinity dimers3. The spread of values measured by SV highlights the difficulty in making accurate measurements at nanomolar protein concentrations, due to dimer-deficient monomers produced by proteolysis or extremely small signal to noise ratios. The similar variance observed in prior work with fluorescence detection AUC suggests that S/N limitations are not the major cause of Kd variations. 1. Jin R, Singh SK, Gu S, Furukawa H, Sobolevsky AI, Zhou J, Jin Y, Gouaux E (2009). EMBO J 28:1812-1823. 2. Clayton A, Siebold C, Gilbert RJ, Sutton GC, Harlos K, McIlhinney RA, Jones EY, Aricescu AR (2009) J Mol Biol 392:1125-1132. 3. Rossmann M, Sukumaran M, Penn AC, Veprintsev DB, Babu MM, Greger IH (2011) EMBO J 30:959-971.

The Multi-zinc Finger Protein ZNF217 Contacts DNA through a Two-finger Domain

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

Binding of Two PriA–PriB Complexes to the Primosome Assembly Site Initiates Primosome Formation

A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage ϕX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA–PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA–PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA–PriB interactions. The effect of PriB is specific for PriA–PAS association, but not for PriA–double-stranded DNA or PriA–single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.

Max-E47, a designed minimalist protein that targets the E-box DNA site in vivo and in vitro.

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nanomolar K(d) values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14, 15, 9, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein

Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem–loop sequence complementary to the transactivation response element, doubly labelled at its 5′ and 3′ ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An “in-house” chemical library of 4800 molecules was screened and five compounds with IC 50 values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.

Effects of CpG Methylation on Recognition of DNA by the Tumour Suppressor p53

Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. Its pattern is altered in cancer and often results in the hypermethylation of the promoter regions and abnormal expression of tumour suppressor genes. Methylation of CpG dinucleotides located in the binding sites of transcription factors may contribute to the development of cancers by preventing their binding or altering their specificity. We studied the effects of CpG methylation on DNA recognition by the tumour suppressor p53, a transcription factor involved in the response to carcinogenic stress. p53 recognises a large number of DNA sequences, many of which contain CpG dinucleotides. We systematically substituted a CpG dinucleotide at each position in the consensus p53 DNA binding sequence and identified substitutions tolerated by p53. We compared the binding affinities of methylated versus non-methylated sequences by fluorescence anisotropy titration. We found that binding of p53 was not affected by cytosine methylation in a majority of cases. However, for a few sequences containing multiple CpG dinucleotides, such as sites in the RB and Met genes, methylation resulted in a four- to sixfold increase in binding of p53. This approach can be used to quantify the effects of CpG methylation on the DNA recognition by other DNA-binding proteins.

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

LicT belongs to a family of bacterial transcriptional antiterminators, which control the expression of sugar-metabolizing operons in response to phosphorylations by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Previous studies of LicT have revealed the structural basis of RNA recognition by the dimeric N-terminal co-antiterminator (CAT) domain on the one hand and the conformational changes undergone by the duplicated regulation domain (PRD1 and PRD2) upon activation on the other hand. To investigate the mechanism of signal transduction between the effector and regulation modules, we have undertaken the characterization of a fragment, including the CAT and PRD1 domains and the linker in-between. Comparative experiments, including RNA binding assays, NMR spectroscopy, limited proteolysis, analytical ultracentrifugation, and circular dichroism, were conducted on native CAT-PRD1 and on a constitutively active CAT-PRD1 mutant carrying a D99N substitution in PRD1. We show that in the native state, CAT-PRD1 behaves as a rather unstable RNA-binding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsin-resistant helix. In the activated mutant form, the CAT-PRD1 linker becomes protease-sensitive, and the helix content decreases, and the CAT module adopts the same dimeric conformation as in isolated CAT, thereby restoring the affinity for RNA. From these results, we propose that a helix-to-coil transition in the linker acts as the structural relay triggered by the regulatory domain for remodeling the effector dimer interface. In essence, the structural mechanism modulating the LicT RNA antitermination activity is thus similar to that controlling the DNA binding activity of dimeric transcriptional regulators.

Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

Damage Control

In the soil bacterium Bacillus subtilis , the checkpoint protein DisA (DNA integrity scanning protein A) scans the bacterial chromosome at the onset of sporulation; it localizes at sites of DNA damage to temporarily block sporulation and allow the bacterium to repair the damage before proceeding. Witte et al . performed structural analysis of Thermotoga maritima DisA and determined that it formed an octameric complex; intriguingly, they found bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) bound to the purified complex. When nucleotide-deprived DisA was crystallized in the presence of Mg 2+ -ATPγS, the crystals were again bound to c-di-AMP, indicating that DisA could act as a diadenylate cyclase. c-di-GMP is known to act as a signaling molecule in bacteria; however, fluorescence anisotropy titration analysis revealed that the binding affinity of fluorescently labeled ATP for both T. maritima and B. subtilis DisA was about 20 times greater than that of fluorescently labeled GTP. Furthermore, nucleotide cyclase assays revealed that both T. maritima and B. subtilis DisA converted ATP to c-di-AMP but did not convert GTP to c-di-GMP. DisA thus appears to be the first protein identified with specific diadenylate cyclase activity. Although neither single-stranded nor double-stranded DNA affected DisA cyclase activity, branched DNA bound to DisA and inhibited its cyclase activity. The authors thus propose that DisA uses c-di-AMP to signal the existence of structures that would interfere with chromosomal segregation, allowing c-di-AMP to join the list of naturally occurring purine-based signaling nucleotides. G. Witte, S. Hartung, K. Büttner, K.-P. Hopfner, Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Mol. Cell 30 , 167-178 (2008). [PubMed]

Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA

The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution of a consensus sequence, we defined the DNA sequence with the highest affinity for full-length p53 and quantified the effects of deviation from it on the strength of protein–DNA interaction. The contributions of individual nucleotides were to a first approximation independent and additive. But, in some cases we observed significant deviations from additivity. Based on affinity data, we constructed a binding predictor that mirrored the existing p53 consensus sequence definition. We used it to search for high-affinity binding sites in the genome and to predict the effects of single-nucleotide polymorphisms in these sites. Although there was some correlation between the Kd and biological function, the spread of the Kds by itself was not sufficient to explain the activation of different pathways by changes in p53 concentration alone.

Equilibrium Analysis of the DNA Binding Domain of the Ultraspiracle Protein Interaction with the Response Element from the hsp27 Gene Promoter—the Application of Molecular Beacon Technology

Ecdysteroids initiate molting and metamorphosis in insects via a receptor which belongs to the superfamily of nuclear receptors. The ecdysone receptor consists of two proteins: the ecdysone receptor (EcR) and the ultraspiracle (Usp). The EcR-Usp dimer conducts transcription through a hsp27(pal) response element. Usp acts as an anchor orienting the whole complex on the DNA. The molecular beacon methodology was applied to detect the sequence-specific DNA of a natural hsp27 (pal) or mutated protein interaction with the DNA binding domain from the Usp. The dissociation constant, K(d), of the UspDBD-hsp27 (pal) complex was determined to be 1.42+/-0.48 nM, whereas K(d) for UspDBD(DeltaA)-hsp27(pal) was 6.6+/-0.5 nM. Mutation of Val-71 for Ala blocks formation of the protein-DNA complex in contrast to Glu-19 mutation for Ala for which K(d)=4.31+/-1.01 nM. The results obtained with the molecular beacon technology are related to those obtained by fluorescence anisotropy titrations.