In vitro selection by using mutated GCN4-bZIP peptides for analysis of peptide-DNA interactions.

In vitro selection has been used as a method to determine the optimal binding site for DNA-binding proteins. We report here in vitro selection of dsDNA sequences that bind to mutated-GCN4-bZIP peptides. The GCN4-bZIP peptide mutated from alanine to histidine on a position-14 that contacts with DNA bound to different sequence from a binding site of wild type peptide.     

Stability of the dimerization domain effects the cooperative DNA binding of short peptides

The basic region peptide derived from the basic leucine zipper protein GCN4 bound specifically to the native GCN4 binding sequences in a dimeric form when the beta-cyclodextrin/adamantane dimerization domain was introduced at the C-terminus of the GCN4 basic region peptide. We describe here how the structure and stability of the dimerization domain affect the cooperative formation of the peptide dimer-DNA complex. The basic region peptides with five different guest molecules were synthesized, and their equilibrium dissociation constants with a peptide possessing beta-cyclodextrin were determined. These values, ranging from 1.3 to 15 microM, were used to estimate the stability of the complexes between the dimers with various guest/cyclodextrin dimerization domains and GCN4 target sequences. An efficient cooperative formation of the dimer complexes at the GCN4 binding sequence was observed when the adamantyl group was replaced with the norbornyl or noradamantyl group, but not with the cyclohexyl group that formed a beta-cyclodextrin complex with a stability that was 1 order of magnitude lower than that of the adamantyl group. Thus, cooperative formation of the stable dimer-DNA complex appeared to be effected by the stability of the dimerization domain. For the peptides that cooperatively formed dimer-DNA complexes, there was no linear correlation between the stability of the inclusion complex and that of the dimer-DNA complex. With the beta-cyclodextrin/adamantane dimerization domain, the basic region peptide dimer preferred to bind to a palindromic 5'-ATGACGTCAT-3' sequence over the sequence lacking the central G.C base pair and that with an additional G.C base pair in the middle. Changing the adamantyl group into a norbornyl group did not alter the preferential binding of the peptide dimers to the palindromic sequence, but slightly affected the selectivity of the dimer for other nonpalindromic sequences. The helical contents of the peptides in the DNA-bound dimer with the adamantyl group were decreased by reducing the stability of the dimer-DNA complex, which was possibly caused by deformation of the helical structure proximal to the dimerization domain.     

Mass spectrometric study of the Escherichia coli repressor proteins, IcIR and GcIR, and their complexes with DNA

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-like proteins, now known as the IclR family, which can be identified by a conserved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCl) for solubility. With the advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordinary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experiment, we found that IclR protein selected from a random pool a sequence which matched exactly that of the presumed binding region of the GclR protein, although IclR does not regulate the gcl gene. We designed a 29 bp synthetic DNA to which IclR and GclR bind, and with which we were able to form noncovalent DNA-protein complexes for further mass spectrometry analysis. These complexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein monomer : dsDNA) = (4 : 1).     

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.     

Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures

Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double-stranded DNA (dsDNA) but not to bind single-stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413-421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity-mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence-specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau-DNA complexes studied.     

Electrospray ionization-mass spectrometry study of the interaction of cisplatin-adducted oligonucleotides with human XPA minimal binding domain protein.

Nucleotide excision repair (NER) is the process responsible for eliminating most ultraviolet (UV) radiation damage from DNA, as well as base alterations caused by a variety of mutagens. The xeroderma pigmentosum group A complementing protein (XPA) is believed to be involved in the early step of NER by recognizing and binding damaged DNA. Recent work has suggested that electrospray ionization-mass spectrometry (ESI-MS) can be an effective tool for the study of protein-DNA complexes. We have used ESI-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to examine the cisplatin-adducted oligonucleotide and its interaction with the human XPA minimal binding domain (XPA-MBD). High-resolution FTICR experiments of the binding products showed that both double-stranded damaged 20-mer and double-stranded undamaged 20-mer formed 1:1 noncovalent complexes with XPA-MBD. A 2:1 binding stoichiometry complex was also observed between XPA-MBD and double-stranded damaged 20-mer. Competitive binding experiments indicated only slightly preferential binding of XPA-MBD with the double-stranded damaged 20-mer compared to the undamaged 20-mer. The results demonstrate that ESI-FTICR mass spectrometry provides a fast and efficient approach for characterizing weak protein-DNA interactions such as the binding between XPA-MBD and a 20-mer oligonucleotide system.     

Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation

IHF and HU are small basic proteins of eubacteria that bind as homodimers to double-stranded DNA and bend the duplex to promote architectures required for gene regulation. These architectural proteins share a common alpha/beta fold but exhibit different nucleic acid binding surfaces and distinct functional roles. With respect to DNA-binding specificity, for example, IHF is sequence specific, while HU is not. We have employed Raman difference spectroscopy and gel mobility assays to characterize the molecular mechanisms underlying such differences in DNA recognition. Parallel studies of solution complexes of IHF and HU with the same DNA nonadecamer (5' --> 3' sequence: TC TAAGTAGTTGATTCATA, where the phage lambda H1 consensus sequence of IHF is underlined) show the following. (i) The structure of the targeted DNA site is altered much more dramatically by IHF than by HU binding. (ii) In the IHF complex, the structural perturbations encompass both the sugar-phosphate backbone and the bases of the consensus sequence, whereas only the DNA backbone is altered by HU binding. (iii) In the presence of excess protein, complexes of order higher than 1 dimer per duplex are detected for HU:DNA, though not for IHF:DNA. The results differentiate structural motifs of IHF:DNA and HU:DNA solution complexes, provide Raman signatures of prokaryotic sequence-specific and nonspecific recognition, and suggest that the architectural role of HU may involve the capability to recruit additional binding partners to even relatively short DNA sequences.     

Quantitative detection of binding of PCNA protein to DNA strands on a 27 MHz quartz-crystal microbalance.

We report here binding kinetics of proliferating cell nuclear antigen (PCNA) to the dsDNA immobilized on a 27 MHz quartz-crystal microbalance. PCNA is a toroidal-shaped protein and encircles dsDNA sliding along the strand. The binding behavior of this sliding clamp protein was compared with those of other side binding proteins such as GCN4-bZIP and Zn-finger peptides.     

The interaction of the A and A* proteins of bacteriophage phi X174 with single-stranded and double-stranded phi X DNA in vitro

The binding of the bacteriophage phi X 174-coded A and A* proteins to single-stranded (ssDNA) and double-stranded (dsDNA ) phi X DNA was studied by electron microscopy. The interaction of the A* protein with ssDNA and dsDNA was also studied by sedimentation velocity centrifugation. It was shown that the binding of the A and A* proteins to ssDNA occurs in a non-cooperative manner and requires no or very little sequence specificity under the conditions used here. Both protein-ssDNA complexes have the same compact structure caused by intrastrand cross-linking through the interaction of protein molecules with separate parts of the ssDNA molecule. The A protein does not bind to phi X dsDNA in the absence of divalent cations. The A* protein does bind to dsDNA, although it has a strong preference for binding to ssDNA. The structure of the A* protein-dsDNA complexes is different from that of the A* protein-ssDNA complexes, as the former have a rosette-like structure caused by protein-protein interactions. High ionic strengths favour the formation of large condensed aggregates.     

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein–protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.     

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.     

Kinetic analysis of specificity of duplex DNA targeting by homopyrimidine peptide nucleic acids

A simple theoretical analysis shows that specificity of double-stranded DNA (dsDNA) targeting by homopyrimidine peptide nucleic acids (hpyPNAs) is a kinetically controlled phenomenon. Our computations give the optimum conditions for sequence-specific targeting of dsDNA by hpyPNAs. The analysis shows that, in agreement with the available experimental data, kinetic factors play a crucial role in the selective targeting of dsDNA by hpyPNAs. The selectivity may be completely lost if PNA concentration is too high and/or during prolonged incubation of dsDNA with PNA. However, quantitative estimations show that the experimentally observed differences in the kinetic constants for hpyPNA binding with the correct and mismatched DNA sites are sufficient for sequence-specific targeting of long genomic DNA by hpyPNAs with a high yield under appropriate experimental conditions. Differential dissociation of hpyPNA/dsDNA complexes is shown to enhance the selectivity of DNA targeting by PNA.     

An Integrative Approach Combining Noncovalent Mass Spectrometry, Enzyme Kinetics and X-ray Crystallography to Decipher Tgt Protein-Protein and Protein-RNA Interaction

The tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used (i) to confirm the dimeric oligomerization state of Tgt in solution and (ii) to evidence the binding stoichiometry of the complex formed between Tgt and its full-length substrate tRNA. To further investigate the importance of Tgt protein-protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X-ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt:inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein-protein interaction, which could provide a new way to inhibit Tgt.     

The effect of C-terminal helix stabilization on specific DNA binding by monomeric GCN4 peptides.

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.     

PNA zipper as a dimerization tool: Development of a bZip mimic

The article describes the use of a PNA duplex (PNA zipper) as a tool to dimerize or bring in close proximity two polypeptides or protein domains. The amino acid sequence to be dimerized is covalently bound to complementary PNA sequences. Annealing of the PNA strands results in dimer formation. To test the ability of the “PNA‐zipper” as a dimerization tool, we designed a GCN4 mimetic, where the leucine‐zipper dimerization domain was replaced by the PNA zipper, whereas the basic DNA‐binding domain was covalently attached to the PNA. The molecule was assembled by chemical ligation of the peptide corresponding to the DNA‐binding domain of GCN4 modified with a succinyl thioester with two complementary PNAs harboring a cysteine residue. Electromobility‐shift experiments show the ability of the PNA zipper‐GCN4 to bind selected DNA duplexes. The PNA zipper‐GCN4 binds both the TRE and CRE DNA sites, but it does not bind TRE and CRE mutants containing even a single base mutation, as the native GCN4. The ability to fold upon complexation with DNA was investigated by CD. A good correlation between the ability of the PNA zipper‐GCN4 to fold into α helices and the ability to bind DNA was found. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 434–441, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com