Cooperative Protein Binding Research Articles

Conditional Cooperativity in DNA Minor-Groove Recognition by Oligopeptides.

The recognition of specific DNA sequences in processes such as transcription is associated with a cooperative binding of proteins. Some transcription regulation mechanisms involve additional proteins that can influence the binding cooperativity by acting as corepressors or coactivators. In a conditional cooperativity mechanism, the same protein can induce binding cooperativity at one concentration and inhibit it at another. Here, we use calorimetric (ITC) and spectroscopic (UV, CD) experiments to show that such conditional cooperativity can also be achieved by the small DNA-directed oligopeptides distamycin and netropsin. Using a global thermodynamic analysis of the observed binding and (un)folding processes, we calculate the phase diagrams for this system, which show that distamycin binding cooperativity is more pronounced at lower temperatures and can be first induced and then reduced by increasing the netropsin or/and Na+ ion concentration. A molecular interpretation of this phenomenon is suggested.

Steady-state fluctuations of a genetic feedback loop with fluctuating rate parameters using the unified colored noise approximation

A common model of stochastic auto-regulatory gene expression describes promoter switching via cooperative protein binding, effective protein production in the active state and dilution of proteins. Here we consider an extension of this model whereby colored noise with a short correlation time is added to the reaction rate parameters—we show that when the size and timescale of the noise is appropriately chosen it accounts for fast reactions that are not explicitly modeled, e.g., in models with no mRNA description, fluctuations in the protein production rate can account for rapid multiple stages of nuclear mRNA processing which precede translation in eukaryotes. We show how the unified colored noise approximation can be used to derive expressions for the protein number distribution that is in good agreement with stochastic simulations. We find that even when the noise in the rate parameters is small, the protein distributions predicted by our model can be significantly different than models assuming constant reaction rates.

Directional allosteric regulation of protein filament length.

Cofilin and ADF are cytoskeleton remodeling proteins that cooperatively bind and fragment actin filaments. Bound cofilin molecules do not directly interact with each other, indicating that cooperative binding of cofilin is mediated by the actin filament lattice. Cofilactin is therefore a model system for studying allosteric regulation of self-assembly. How cofilin binding changes structural and mechanical properties of actin filaments is well established. Less is known about the interaction energies and the thermodynamics of filament fragmentation, which describes the collective manner in which the cofilin concentration controls mean actin filament length. Here, we provide a general thermodynamic framework for allosteric regulation of self-assembly, and we use the theory to predict the interaction energies of experimental actin filament length distributions over a broad range of cofilin binding densities and for multiple cofilactin variants. We find that bound cofilin induces changes in nearby actin-actin interactions, and that these allosteric effects are propagated along the filament to affect up to four neighboring cofilin-binding sites (i.e., beyond nearest-neighbor allostery). The model also predicts that cofilin differentially stabilizes and destabilizes longitudinal versus lateral actin-actin interactions, and that the magnitude, range, asymmetry, and even the sign of these interaction energies can be altered using different actin and cofilin mutational variants. These results demonstrate that the theoretical framework presented here can provide quantitative thermodynamic information governing cooperative protein binding and filament length regulation, thus revealing nanometer length-scale interactions from micron length-scale "wet-lab" measurements.

Cooperative DNA binding by proteins through DNA shape complementarity.

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein–protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis β-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.

Crazy About CRISPR: An Interview with Francisco Mojica.

Crazy About CRISPR: An Interview with Francisco Mojica.

Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor α and Specificity Protein 1 with Composite DNA Elements.

Estrogen receptor α (ERα) and Specificity protein 1 (Sp1) are transcription factors (TF) that are involved in regulating progesterone receptor (PR) gene expression through cooperative interactions with DNA. The natural composite DNA +571 ERE/Sp1 site in promoter A of the progesterone receptor contains a half-site of estrogen response elements (½ERE) upstream of two Sp1 binding sites (the proximal Sp1 (Sp1/P) and distal Sp1 (Sp1/D)) with a 4 bp spacer. Here, we have developed a protocol for studying the cooperative interaction of Sp1 and ERα with the composite DNA of +571 ERE/Sp1 site using Biacore T200, a high sensitivity surface plasmon resonance spectroscopy. With this protocol, we have concluded that Sp1 binding enhances the overall ERα binding to the composite DNA. We have also determined the optimal spacer distance between the ½ERE and Sp1/D for the best cooperative protein binding. This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor-DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators.

Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes.

Electrophoretic mobility shift assays (EMSA) have proven their usefulness for studying interactions between biological molecules. In the present protocol, a purified protein of interest is mixed with a 5'-end radiolabeled DNA probe. The bound complexes are separated by electrophoretic migration through a polyacrylamide gel and detected with a phosphorimager. The applications of EMSA are diverse, from thermodynamic and kinetic analyses to observation of bending and other conformational changes, stoichiometric inferences, or insights into cooperative protein binding.

Effects of molecular noise on bistable protein distributions in rod-shaped bacteria.

The distributions of many proteins in rod-shaped bacteria are far from homogeneous. Often they accumulate at the cell poles or in the cell centre. At the same time, the copy number of proteins in a single cell is relatively small making the patterns noisy. To explore limits to protein patterns due to molecular noise, we studied a generic mechanism for spontaneous polar protein assemblies in rod-shaped bacteria, which are based on cooperative binding of proteins to the cytoplasmic membrane. For mono-polar assemblies, we find that the switching time between the two poles increases exponentially with the cell length and with the protein number. This feature could be beneficial to organelle maintenance in ageing bacteria.

HU Protein Induces Incoherent DNA Persistence Length

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding.

Model of DNA Bending by Cooperative Binding of Proteins

We present a model of nonspecific cooperative binding of proteins to DNA in which the binding of isolated proteins generates local bends but binding of proteins at neighboring sites on DNA straightens the polymer. We solve the statistical mechanical problem and calculate the effective persistence length, site occupancy, and cooperativity. Cooperativity leads to nonmonotonic variation of the persistence length with protein concentration, and to an unusual shape of the binding isotherm. The results are in qualitative agreement with recent single molecule experiments on nucleoid protein HU-DNA complexes.

LeuO Protein Delimits the Transcriptionally Active and Repressive Domains on the Bacterial Chromosome

LeuO protein relieves bacterial gene silencer AT8-mediated transcriptional repression as part of a promoter relay mechanism found in the ilvIH-leuO-leuABCD gene cluster. The gene silencing activity has recently been characterized as a nucleoprotein filament initiated at the gene silencer. In this gene locus, the nucleoprotein filament cis-spreads toward the target leuO promoter and results in the repression of the leuO gene. Although the cis-spreading nature of the transcriptionally repressive nucleoprotein filament has been revealed, the mechanism underlying LeuO-mediated gene silencing relief remains unknown. We have demonstrated here that LeuO functions analogously to the eukaryotic boundary element that delimits the transcriptionally active and repressive domains on the chromosome by blocking the cis-spreading pathway of the transcriptionally repressive heterochromatin. Given that one LeuO-binding site is positioned between the gene silencer and the target promoter, the simultaneous presence of a second LeuO-binding site synergistically enhances the blockade, resulting in a cooperative increase in LeuO-mediated gene silencing relief. A known DNA loop-forming protein, the lac repressor (LacI), was used to confirm that cooperative protein binding via DNA looping is responsible for the blocking synergy. Indeed, a distal LeuO site located downstream cooperates with the LeuO sites located upstream of the leuO gene, resulting in synergistic relief for the repressed leuO gene via looping out the intervening DNA between LeuO sites in the ilvIH-leuO-leuABCD gene cluster.

LAST motifs and SMART domains in gene 32 protein: an unfolding story of autoregulation?

Bacteriophage T4 gene 32 protein is a classical single strand-specific DNA binding protein. It is a single polypeptide chain of 301 amino acid residues that consists of three structural domains, each of which has a binding function. The N-terminal domain is involved in homotypic protein-protein interaction (the basis of binding cooperativity), the core domain binds single strands directly, and the C-terminal domain has a role in heterotypic protein-protein association. The three domains have traditionally been thought to be independent of each other. However, the observation of a striking repetition of a basic, polar sequence (the "LAST" Motif), seen in both the N-terminal and core domains, suggests a linkage between these domains. Moreover, the C-domain and adjoining portion (flap) of the core are highly acidic, and are potential mimics of single-stranded DNA. With these observations, I construct a model in which this flap is associated with the ssDNA binding site in the absence of DNA, and upon cooperative protein binding to DNA, the flap now associates with the N-terminal domain of the adjacent DNA-bound protein. The flap thus acts as a gate, which might slow the binding of the protein to DNA. This could lead to the regulation of the protein's various interactions with other proteins, as well as affect its ability to lower DNA melting temperature.

Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA.

An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly.

Protein-protein interactions in a higher-order structure direct lambda site-specific recombination

The highly directional site-specific recombination of bacteriophage lambda is tightly regulated by the binding of three different proteins to a complex array of sites. The manner in which these reactions are both stimulated and inhibited by co-operative binding of proteins to specific sites on the P arm of attP and attR has been elucidated by correlation of nuclease protection with recombination studies of both wild-type and mutant DNAs. In addition to co-operative forces, there is a specific competitive interaction that allows the protein-DNA complex to serve as a “biological switch”. This switch does not depend upon the simple occlusion of DNA binding sites by neighboring proteins; but, rather, the outcome of this competition is dependent on long-range interactions that vary between the higher-order structures of attP and attR. These higher-order structures are dependent on co-operative interactions involving three proteins binding to five or more sites.

DNA "melting" proteins. III. Fluorescence "mapping" of the nucleic acid binding site of bacteriophage T4 gene 32-protein.

The intrinsic tryptophan fluorescence of bacteriophage T4-coded gene 32-protein is found to be partially quenched on binding a variety of mono-, oligo-, and polynucleotides. This phenomenon is exploited to partially "map" the nucleic acid binding site of the protein. The intrinsic fluorescence spectrum of the protein peaks at about 347 nm, compared to 359 nm for the fully solvated model fluorophore, N-acetyl-L-tryptophanamide. Nucleotide binding, or collisional quenching by iodide ion, reduces the intensity of the fluorescence, with little or no peak shift. Small ligands, ranging in size from ribose- and deoxyribose-phosphate to tetranucleotides, quench the fluorescence by 2 to 6%; larger ligands quench from 20 to 35% of the intrinsic protein fluorescence. Iodide quenching experiments subjected to Stern-Vollmer analysis suggest that the binding of short nucleotide-containing ligands brings about a conformational change in the protein, fully exposing a tryptophan side chain to the solvent environment. The fluorescence of this tryptophan is fully quenched by the binding of d(Ap)2, but is largely unaffected by the binding of d(ApA) or d(pA)2, indicating both that this (tryptophan) "reporter" residue is located in the nucleic acid binding site and that binding is polar, i.e. polynucleotide chains of only one orientation are complexed. Long oligonucleotides fully quench the fluorescence of this binding site tryptophan. At high salt concentration (2 M NaCl), gene 32-protein forms self-limited dimers (Carroll, R.B., Neet, K.E., and Goldthwait, D.A. (1972) Proc. Natl, Acad. Sci. U.S.A. 69, 2741-2744; (1975) J. Mol. Biol. 91, 275-291). These dimers, in either high salt or in low salt after cross-linking, fail to bind nucleotides, suggesting that dimer formation partially occludes the nucleic acid binding site and thus that these dimers are probably not involved as intermediates in cooperative protein binding to the DNA. On the other hand, dimerization apparently results in a conformational change which fully exposes the "reporter" tryptophan to iodide quenching. These results are used to formulate a model of some of the nucleic acid-protein and protein-protein interactions involved in the cooperative binding of gene 32-protein to single-stranded DNA.