The recognition of a noncanonical RNA base pair by a zinc finger protein.

BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.     

Correlation of the side-chain hubs with the functional residues in DNA binding protein structures

The structure networks of DNA-binding proteins have been constructed and analyzed. The detailed analysis of the networks indicates a strong relation between the positions of the residues interacting with DNA and those that form extensive interactions within the protein structure (called hubs). This study shows that the functional residues in these proteins are held in place by efficient scaffolding of the structure using side-chain interactions, thus highlighting the role of these side-chain hubs with respect to the functional residues in the protein structure.     

Role of protein structure and the role of individual fingers in zinc finger protein–DNA recognition: a molecular dynamics simulation study and free energy calculations

Molecular dynamics and MM_GBSA energy calculations on various zinc finger proteins containing three and four fingers bound to their target DNA gave insights into the role of each finger in the DNA binding process as part of the protein structure. The wild type Zif 268 (PDB code: 1AAY) gave a ΔG value of ??76.1 (14) kcal/mol. Zinc fingers ZF1, ZF2 and ZF3 were mutated in one experiment and in another experiment one finger was cut and the rest of the protein was studied for binding. The ΔΔG values for the Zinc Finger protein with both ZF1 and ZF2 mutated was +?80 kcal/mol, while mutating only ZF1 the ΔΔG value was +?52 kcal/mol (relative to the wild type). Cutting ZF3 and studying the protein consisting only of ZF1 linked to ZF2 gave a ΔΔG value of +?68 kcal/mol. Upon cutting ZF1, the resulting ZF2 linked to ZF3 protein gave a ΔΔG value of +?41 kcal/mol. The above results shed light on the importance of each finger in the binding process, especially the role of ZF1 as the anchoring finger followed in importance by ZF2 and ZF3. The energy difference between the binding of the wild type protein Zif268 (1AAY) and that for individual finger binding to DNA according to the formula: ΔΔG_{linkers, otherstructuralfactors}?=?ΔG_zif268???(ΔG_F1+F2+F3) gave a value?=???44.5 kcal/mol. This stabilization can be attributed to the contribution of linkers and other structural factors in the intact protein in the DNA binding process. DNA binding energies of variant proteins of the wild type Zif268 which differ in their ZF1 amino acid sequence gave evidence of a good relationship between binding energy and recognition and specificity, this finding confirms the reported vital role of ZF1 in the ZF protein scanning and anchoring to the target DNA sequence. The role of hydrogen bonds in both specific and nonspecific amino acid-DNA contacts is discussed in relation to mutations. The binding energies of variant Zinc Finger proteins confirmed the role of ZF1 in the recognition, specificity and anchoring of the zinc finger protein to DNA.     

基因调控中的DNA结合蛋白

DNA结合蛋白通过与特定位点的DNA结合可进行基因功能的调控。根据蛋白质结晶学和分子生物学研究了蛋白质-DNA之间的相互作用。对几种DNA结合域,如“螺旋-转折-螺旋”、“亮氮酸拉链”、“锌指”进行了讨论。     

Protein-Controlled Actuation of Dynamic Nucleic Acid Networks by Using Synthetic DNA Translators**

Integrating dynamic DNA nanotechnology with protein-controlled actuation will expand our ability to process molecular information. We have developed a strategy to actuate strand displacement reactions using DNA-binding proteins by engineering synthetic DNA translators that convert specific protein-binding events into trigger inputs through a programmed conformational change. We have constructed synthetic DNA networks responsive to two different DNA-binding proteins, TATA-binding protein and Myc-Max, and demonstrated multi-input activation of strand displacement reactions. We achieved protein-controlled regulation of a synthetic RNA and of an enzyme through artificial DNA-based communication, showing the potential of our molecular system in performing further programmable tasks.     

DIFFERENTIAL EXPRESSION OF PYRIMIDINE DIMER-BINDING PROTEINS IN NORMAL AND UV LIGHT-TREATED VERTEBRATE CELLS

Abstract— The expression of UV damage-specific DNA-binding proteins was examined in various phylogenetically distant species with differing DNA repair phenotypes. Two distinct constitutive DNA-binding activities, one specific for cyclobutane pyrimidine dimers and the other for non-cyclobutane dimer photoproducts, were detected. The expression of these binding activity was found to be variable throughout the animal kingdom: cold-blooded vertebrastes show a constitutive cyclobutance dimer-binding activity excusively, and primates reveal only non-cyclobutane expression (rather than the constitutive presence)of these UV damage-specific DNA-binding activities after UV traeatment correlate with the cell's capacity for DNA repair. In addition, cyclobutane pyrimidine dimer-binding activities could be detected only in cells with eestablished photoreactivating activity     

Physics of engineered protein hydrogels

Artificially engineered proteins and synthetic polypeptides have attracted widespread interest as building blocks for polymer hydrogels. The biophysical properties of the proteins, such as molecular recognition abilities, folded chain structures, and sequence-dependent thermodynamic behavior, enable advances in functional, responsive, and tunable gels. This review discusses the design of polymer hydrogels that incorporate protein domains, highlighting new challenges in polymer physics that are presented by this emerging class of materials. Five types of engineered protein hydrogels are discussed: (a) physically associating protein polymer gels, (b) amorphous artificially engineered protein networks, (c) engineered proteins with crystalline domains, (d) stretchable protein tertiary structures in gels, and (e) protein gels with biological recognition properties. The physics of the protein component and the physical properties of the resulting hydrogels are summarized, illustrating how advances in understanding these systems are leading to exciting novel biofunctional hydrogels. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

Engineering temperature-sensitive SH3 domains.

BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks. SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences. RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive. Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature. Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins. Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells. CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo. Such mutants will be valuable in dissecting SH3-mediated signaling pathways. Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions.     

Sequence specific alkylation of DNA by hairpin pyrrole-imidazole polyamide conjugates

BACKGROUND: Pyrrole-imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification. RESULTS: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the gamma turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide-CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation. CONCLUSIONS: The ability of polyamide-CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms.     

A polyplex qPCR-based binding assay for protein-DNA interactions

The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.     

Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair

Replication protein A (RPA), eukaryotic single-stranded DNA-binding protein, is a key player in multiple processes of DNA metabolism including DNA replication, recombination and DNA repair. Human RPA composed of subunits of 70-, 32- and 14-kDa binds ssDNA with high affinity and interacts specifically with multiple proteins. The RPA heterotrimer binds ssDNA in several modes, with occlusion lengths of 8–10, 13–22 and 30 nucleotides corresponding to global, transitional and elongated conformations of protein. Varying the structure of photoreactive DNA, the intermediates of different stages of DNA replication or DNA repair were designed and applied to identify positioning of the RPA subunits on the specific DNA structures. Using this approach, RPA interactions with various types of DNA structures attributed to replication and DNA repair intermediates were examined. This review is dedicated to blessed memory of Prof. Alain Favre who contributed to the development of photoreactive nucleotide derivatives and their application for the study of protein–nucleic acids interactions.     

Chemical approaches untangling sequence-specific DNA binding by proteins

Structure-based design of novel DNA-binding proteins provides an ultimate test of our understanding of protein-DNA interactions. A combination of synthetic, organic, biochemical and molecular biological approaches has been developed to study the principle of molecular recognition associated with the protein-DNA interactions. The strategies enabled a specific formation of noncovalent peptide dimers and determination of the preferential DNA-binding sequence of short peptides.     

Structure and dynamics of nano-sized raft-like domains on the plasma membrane

Cell membranes are constitutively composed of thousands of different lipidic species, whose specific organization leads to functional heterogeneities. In particular, sphingolipids, cholesterol and some proteins associate among them to form stable nanoscale domains involved in recognition, signaling, membrane trafficking, etc. Atomic-detail information in the nanometer/second scale is still elusive to experimental techniques. In this context, molecular simulations on membrane systems have provided useful insights contributing to bridge this gap. Here we present the results of a series of simulations of biomembranes representing non-raft and raft-like nano-sized domains in order to analyze the particular structural and dynamical properties of these domains. Our results indicate that the smallest (5 nm) raft domains are able to preserve their distinctive structural and dynamical features, such as an increased thickness, higher ordering, lower lateral diffusion, and specific lipid-ion interactions. The insertion of a transmembrane protein helix into non-raft, extended raft-like, and raft-like nanodomain environments result in markedly different protein orientations, highlighting the interplay between the lipid-lipid and lipid-protein interactions.     

A label-free electrochemical test for DNA-binding activities of tumor suppressor protein p53 using immunoprecipitation at magnetic beads

In this paper we extend the application area of the label-free structure-sensitive electrochemical DNA sensing with mercury-based electrodes which is for the first time used, in combination with immunoprecipitation at magnetic beads (MB), for the probing of DNA interactions with tumor suppressor protein p53. The technique relies on capture of the p53-DNA complexes at MB via anti-p53 antibodies, followed by salt-induced dissociation of linear DNA from the complex and its voltammetric detection. Competitive binding of p53 to various plasmid DNA substrates, including lin or scDNAs with or without a specific target site, can easily be followed by ex situ electrochemical analysis of DNA recovered from the immunoprecipitated complexes. Compared to gel electrophoresis which is usually applied to analyze different plasmid DNA forms and their complexes with proteins, the electrochemical detection is faster and allows simpler quantitation of DNA containing free ends at submicrogram levels. We demonstrate applicability of the proposed technique to monitor different DNA-binding activities of wild type and mutant p53 proteins.