Cavities in protein–DNA and protein–RNA interfaces

An analysis of cavities present in protein–DNA and protein–RNA complexes is presented. In terms of the number of cavities and their total volume, the interfaces formed in these complexes are akin to those in transient protein–protein heterocomplexes. With homodimeric proteins protein–DNA interfaces may contain cavities involving both the protein subunits and DNA, and these are more than twice as large as cavities involving a single protein subunit and DNA. A parameter, cavity index, measuring the degree of surface complementarity, indicates that the packing of atoms in protein–protein/DNA/RNA is very similar, but it is about two times less efficient in the permanent interfaces formed between subunits in homodimers. As within the tertiary structure and protein–protein interfaces, protein–DNA interfaces have a higher inclination to be lined by β-sheet residues; from the DNA side, base atoms, in particular those in minor grooves, have a higher tendency to be located in cavities. The larger cavities tend to be less spherical and solvated. A small fraction of water molecules are found to mediate hydrogen-bond interactions with both the components, suggesting their primary role is to fill in the void left due to the local non-complementary nature of the surface patches.     

UPA, a universal protein array system for quantitative detection of protein–protein, protein–DNA, protein–RNA and protein–ligand interactions

Protein–protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein–protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.     

Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5′-{N}N₁N₂N₃N₄N₅N₆{N}-3′ consisted of a fixed hexamer motif N₁N₂N₃N₄N₅N₆ in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase–ssDNA complexes with the temperature increasing from 0°C to 50°C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5′-NNG(A/T/C)GNN-3′ with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.     

Detection of protein–DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA–protein interaction

A simple, direct method for the detection of DNA–protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purβ (mPurβ), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurβ and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurβ were 25 and 3 μg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

A novel strategy for the identification of protein–DNA contacts by photocrosslinking and mass spectrometry

Photochemical crosslinking is a method for studying the molecular details of protein–nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe³⁺-IMAC (immobilized metal affinity chromatography) purification of peptide–oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein–DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide–oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe³⁺-IMAC. A combination of MS analysis of the peptide–nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys²⁰⁹ of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys²⁰⁹ is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.     

Rapid parallel mutation scanning of gene fragments using a microelectronic protein–DNA chip format

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.     

Semisynthesis and optimization of G protein‐coupled receptor mimics

We have recently developed a soluble mimic of the corticotropin‐releasing factor receptor type 1 (CRF1), a membrane‐spanning G protein‐coupled receptor, which allowed investigations on receptor–ligand interactions. The CRF1 mimic consists of the receptor N‐terminus and three synthetic extracellular loops (ECL1–3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost‐efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop‐mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A novel approach for the identification of protein–protein interaction with integral membrane proteins

Protein–protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein–protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein–protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein–protein interactions with high specificity and selectivity, where other methods fail.     

Individual-site binding data and the energetics of protein–DNA interactions

Individual-site isotherms for the binding of bacteriophage λ repressor to the left and right λ operators have been determined [D. F. Senear, M. Brenowitz, M. A. Shea, and G. K. Ackers (1986) Biochemistry, Vol. 25, pp. 7344–7354.] using the DNAse protection technique [ footprinting; D. J. Galas and A. Schmitz (1978) Nucleic Acids Research, Vol. 5, pp. 3157–3170]. These extensive data have been interpreted with a quantitative model that emphasized cooperative interactions between adjacently bound ligands [occupied ? occupied interactions; G. K. Ackers, A. D. Johnson, and M. A. Shea (1982) Proceedings of the National Academy of Science, USA, Vol. 79, pp. 1129–1133]. Overlooked in this model are the effects of cooperative interactions between a site containing a bound ligand and its neighboring unoccupied site (occupied ? unoccupied interactions). This paper reinterprets the existing data with a model that considers occupied ? unoccupied as well as occupied ? occupied interactions. The results yield parameters that differ substantially from those already reported. A discussion on the advisability of ignoring occupied ? unoccupied interactions is included. © 1993 John Wiley & Sons, Inc.     

Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein–DNA footprinting

Nucleic acid fragmentation (footprinting) by ·OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid–protein interactions. This method has proven valuable because it provides structural information with single base pair resolution. Recent developments in the field introduced the ‘synchrotron X-ray footprinting’ method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds. We developed a complementary method that utilizes X-rays generated from a conventional rotating anode machine in which nucleic acid footprints can be generated by X-ray exposures as short as 100–300 ms. Our theoretical and experimental studies indicate that efficient cleavage of nucleic acids by X-rays depends upon sample preparation, energy of the X-ray source and the beam intensity. In addition, using this experimental set up, we demonstrated the feasibility of conducting X-ray footprinting to produce protein–DNA protection portraits at sub-second timescales.     

Effect of DNA local conformation on the affinity and binding specificity of bis-netropsins to DNA

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group (<--Nt-Pt(NH3)-Nt-->) or aliphatic pentamethylene chain (<--Nt-(CH2)5-Nt-->), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of <--Nt-(CH2)5-Nt--> with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for <--Nt-Pt(NH3)-Nt--> and <--Nt-(CH2)5-Nt--> binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.     

Diffusion-controlled protein–DNA association: Influence of segemental diffusion of the DNA

The intrachain reaction theory of Wilemki and Fixman [(1974) J. Chem. Phys. 60 , 866–877] is used to assess the influence of internal DNA motions on various protein–DNA association schemes. It is found that, for large proteins, the diffusional association rate can be totally dominated by these motions rather than by the free-trans-lational diffusion rates. Also, the time required for the diffusion together of two DNA segments is estimated. This estimate can be used to provide an upper limit for the rate of intrachain cyclization, and also for the effective intrachain transfer rate of a protein bound to a DNA chain.     

Vacancy affinity capillary electrophoresis to study competitive protein–drug binding

The suitability of the vacancy affinity capillary electrophoretic method (VACE) for study of displacement of a target drug from a protein by simultaneously administered drugs was investigated. As test system, the displacement of warfarin from bovine serum albumin (BSA) by furosemide and phenylbutazone was selected. It appears that the displacement can be observed well from the shift of the actual mobility of warfarin when a displacer drug is added. Also, the competitive action of the displacer drugs (affinity for BSA) is clearly visible. The VACE method seems to be attractive for rapid assesssment of information about the competitive properties of coadministered compounds.