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1.  Effect of Electrostatics on Aggregation of Prion Protein Sup35 Peptide 
Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic Force Microscopy (AFM) and Thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI=5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0) suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by the experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 hours, whereas the lag time decreases ~5 times when ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 hours under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. Ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics on dissociation of transient peptide dimers.
PMCID: PMC3482402  PMID: 22466073
AFM; Force spectroscopy; protein aggregation; electrostatics; nanoimaging
2.  Centromere chromatin: a loose grip on the nucleosome? 
PMCID: PMC3970403  PMID: 24389544
3.  Nanoprobing of the effect of Cu2+ cations on misfolding, interaction and aggregation of amyloid β peptide 
Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer’s disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu2+. Cu2+ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu2+ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu2+ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu2+ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu2+ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu2+ cations induce Aβ42 peptide structural changes resulting in N–termini interactions within the dimers. Cu2+ cations also stabilize the dimers. No effects of Cu2+ cations on Aβ–Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu2+ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu2+ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu2+ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu2+ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu2+.
PMCID: PMC3586772  PMID: 23143330
Amyloid β-protein; Aβ42; Alzheimer’s disease; Cu2+ cations; Single molecule force spectroscopy; Atomic force microscopy imaging
4.  Preparation of DNA and nucleoprotein samples for AFM imaging 
Micron (Oxford, England : 1993)  2010;42(2):196-206.
Sample preparation techniques allowing reliable and reproducible imaging of DNA with various structures, topologies and complexes with proteins are reviewed. The major emphasis is given to methods utilizing chemical functionalization of mica, enabling preparation of the surfaces with required characteristics. The methods are illustrated by examples of imaging of different DNA structures. Special attention is given to the possibility of AFM to image the dynamics of DNA at the nanoscale. The capabilities of time-lapse AFM in aqueous solutions are illustrated by imaging of dynamic processes as transitions of local alternative structures (transition of DNA between H and B forms). The application of AFM to studies of protein-DNA complexes is illustrated by a few examples of imaging site-specific complexes, as well as such systems as chromatin. The time-lapse AFM studies of protein-DNA complexes including very recent advances with the use of high-speed AFM are reviewed.
PMCID: PMC2997872  PMID: 20864349
AFM; surface functionalization; silanes; DNA; DNA supercoiling; alternative DNA structures; protein-DNA complexes; single molecule imaging
5.  Structure and Dynamics of Dinucleosomes Assessed by Atomic Force Microscopy 
Journal of Amino Acids  2011;2012:650840.
Dynamics of nucleosomes and their interactions are important for understanding the mechanism of chromatin assembly. Internucleosomal interaction is required for the formation of higher-order chromatin structures. Although H1 histone is critically involved in the process of chromatin assembly, direct internucleosomal interactions contribute to this process as well. To characterize the interactions of nucleosomes within the nucleosome array, we designed a dinucleosome and performed direct AFM imaging. The analysis of the AFM data showed dinucleosomes are very dynamic systems, enabling the nucleosomes to move in a broad range along the DNA template. Di-nucleosomes in close proximity were observed, but their population was low. The use of the zwitterionic detergent, CHAPS, increased the dynamic range of the di-nucleosome, facilitating the formation of tight di-nucleosomes. The role of CHAPS and similar natural products in chromatin structure and dynamics is also discussed.
PMCID: PMC3268019  PMID: 22312477
6.  Nanoimaging for prion related diseases 
Prion  2010;4(4):265-274.
Misfolding and aggregation of prion proteins is linked to a number of neurodegenerative disorders such as Creutzfeldt-Jacob disease (CJD) and its variants: Kuru, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. In prion diseases, infectious particles are proteins that propagate by transmitting a misfolded state of a protein, leading to the formation of aggregates and ultimately to neurodegeneration. Prion phenomenon is not restricted to humans. There are a number of prion-related diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as “mad cow disease”) in cattle. All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. Prion proteins were also found in some fungi where they are responsible for heritable traits. Prion proteins in fungi are easily accessible and provide a powerful model for understanding the general principles of prion phenomenon and molecular mechanisms of mammalian prion diseases. Presently, several fundamental questions related to prions remain unanswered. For example, it is not clear how prions cause the disease. Other unknowns include the nature and structure of infectious agent and how prions replicate. Generally, the phenomenon of misfolding of the prion protein into infectious conformations that have the ability to propagate their properties via aggregation is of significant interest. Despite the crucial importance of misfolding and aggregation, very little is currently known about the molecular mechanisms of these processes. While there is an apparent critical need to study molecular mechanisms underlying misfolding and aggregation, the detailed characterization of these single molecule processes is hindered by the limitation of conventional methods. Although some issues remain unresolved, much progress has been recently made primarily due to the application of nanoimaging tools. The use of nanoimaging methods shows great promise for understanding the molecular mechanisms of prion phenomenon, possibly leading toward early diagnosis and effective treatment of these devastating diseases. This review article summarizes recent reports which advanced our understanding of the prion phenomenon through the use of nanoimaging methods.
PMCID: PMC3268959  PMID: 20724837
protein misfolding; prion; atomic force microscopy; nanomedicine; force spectroscopy
7.  Assembly Of The SLIP1-SLBP Complex On Histone mRNA Requires Heterodimerization And Sequential Binding Of SLBP Followed By SLIP1 
Biochemistry  2013;52(3):520-536.
The SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high affinity (KD < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has weak affinity (KD ~3 µM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop, followed by association with SLIP1 is required to form an “active” ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced histone mRNA abundance in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo protecting it from proteolytic degradation as an inactive hetero-tetramer, or alternatively, formation of the SLIP1-SLBP hetero-tetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.
PMCID: PMC3580866  PMID: 23286197
MIF4GD; translation initiation; oligomerization; intrinsically disordered protein; phosphorylation; histone; SLBP; SLIP1
8.  Nucleosomes structure and dynamics: effect of CHAPS 
Dynamics of nucleosomes and spontaneous unwrapping of DNA are fundamental property of the chromatin enabling access to nucleosomal DNA for regulatory proteins. Probing of such dynamics of nucleosomes performed by single molecule techniques revealed a large scale dynamics of nucleosomes including their spontaneous unwrapping. Dissociation of nucleosomes at low concentrations is a complicating issue for studies with single molecule techniques. In this paper, we tested the ability of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) to prevent dissociation of nucleosomes. The study was performed with mononucleosome system assembled with human histones H2A, H2B, H3 and H4 on the DNA substrate containing sequence 601 that provides the sequencespecific assembly of nucleosomes. We used Atomic Force Microscopy (AFM) to directly identify nucleosomes and analyze their structure at the nanometer level. These studies showed that in the presence of CHAPS at millimolar concentrations, nucleosomes, even at sub-nanomolar concentrations, remain intact over days compared to a complete dissociation of the same nucleosome sample over 10 min in the absence of CHAPS. Importantly, CHAPS does not change the conformation of nucleosomes as confirmed by the AFM analysis. Moreover, 16 µM CHAPS stabilizes nucleosomes in over one hour incubation in the solution containing as low as 0.4 nM in nucleosomes. The stability of nucleosomes is slightly reduced at physiological conditions (150 mM NaCl), although the nucleosomes dissociate rapidly at 300 mM NaCl. The sequence specificity of the nucleosome in the presence of CHAPS decreased suggesting that the histone core translocates along the DNA substrate utilizing sliding mechanism.
PMCID: PMC3180101  PMID: 21969098
Chromatin; nucleosome structure; AFM; single molecule analysis; DNA; CHAPS
9.  Dynamics of Nucleosomes Revealed by Time-Lapse AFM 
Biochemistry  2009;48(33):7842-7848.
The dynamics of chromatin provide the access to DNA within nucleosomes and therefore, this process is critically involved into the regulation of chromatin function. However, our knowledge on the large-range dynamics of nucleosomes is limited. The questions, such as the range of opening of the nucleosome, and the mechanism whereby the opening occurs and propagates, remain unknown. Here we applied single molecule time lapse AFM imaging to directly visualize the dynamics of nucleosomes and identify the mechanism of the large range DNA exposure. With this technique, we are able to observe the process of unwrapping of nucleosomes. The unwrapping of nucleosomes proceeds from the ends of the particles, allowing for the unwrapping of DNA regions as large as dozens of base pairs. This process may lead to a complete unfolding of nucleosomes and dissociation of the histone core from the complex. The unwrapping occurs in the absence of proteins involved in the chromatin remodeling that require ATP hydrolysis for their function, suggesting that the inherent dynamics of nucleosomes can contribute to the chromatin unwrapping process. These finding shed a new light to molecular mechanisms of nucleosome dynamics and provide novel hypotheses to the understanding of the action of remodeling proteins as well as other intracellular systems in the chromatin dynamics.
PMCID: PMC2752652  PMID: 19618963
10.  Mica Functionalization for Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy 
Methods in molecular biology (Clifton, N.J.)  2013;931:10.1007/978-1-62703-056-4_14.
Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.
PMCID: PMC3848609  PMID: 23027008
Atomic force microscopy; AFM; Mica functionalization; Surface chemistry; Silanes; Silatranes; DNA structure and dynamics; Protein-DNA complexes
11.  Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target 
Biochemistry  2012;51(7):10.1021/bi201863z.
Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that Escherichia coli SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg2+ cations or a high ionic strength. In the absence of Mg2+ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg2+ cations.
PMCID: PMC3848610  PMID: 22304461
12.  AFM for analysis of structure and dynamics of DNA and protein-DNA complexes 
Methods (San Diego, Calif.)  2008;47(3):206-213.
This paper describes protocols for studies of structure and dynamics of DNA and protein-DNA complexes with AFM utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of local and global conformations DNA with the major focus on the time-lapse imaging of DNA in aqueous solutions is illustrated by the study of dynamics of Holliday junctions including branch migration. The analysis of nucleosome dynamics is selected as an example to illustrate the application of the time-lapse AFM to studies of dynamics of protein-DNA complexes. The force spectroscopy is the modality of AFM with a great importance to various fields of biomedical studies. The AFM force spectroscopy approach for studies of specific protein-DNA complexes is illustrated by the data on analysis of dynamics of synaptic SfiI-DNA complexes. When necessary, additional specifics are added to the corresponding example.
PMCID: PMC2667448  PMID: 18835446
Atomic force microscopy (AFM); Force microscopy; DNA dynamics; Local DNA structures; Holliday junctions; DNA–protein interactions; Protein–protein interactions; Single-molecule techniques; Solution time lapse imaging
13.  Mechanism of amyloid β−protein dimerization determined using single−molecule AFM force spectroscopy 
Scientific Reports  2013;3:2880.
Aβ42 and Aβ40 are the two primary alloforms of human amyloid β−protein (Aβ). The two additional C−terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single−molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C−termini, the N−terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N−terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N−terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.
PMCID: PMC3791449  PMID: 24096987
14.  Nanoscale structure and dynamics of ABOBEC3G complexes with single-stranded DNA 
Biochemistry  2012;51(32):6432-6440.
The DNA cytosine deaminase APOBEC3G (A3G) is capable of blocking retrovirus replication by editing viral cDNA and impairing reverse transcription. However, the biophysical details of this host-pathogen interaction are unclear. Here we applied atomic force microscopy (AFM) and hybrid DNA substrates to investigate properties of A3G bound to single-stranded DNA (ssDNA). Hybrid DNA substrates included ssDNA with 5′ or 3′ ends attached to DNA duplexes (tail-DNA) and gap-DNA substrates, in which ssDNA is flanked by two double-stranded fragments. We found that A3G binds with similar efficiency to the 5′ and 3′ substrates, suggesting that ssDNA polarity is not an important factor. Additionally, we observed that A3G binds the single-stranded region of the gap-DNA substrates with the same efficiency as tail-DNA. These results demonstrate that single-stranded DNA ends are not needed for A3G binding. The protein stoichiometry does not depend on the ssDNA substrate type, but the ssDNA length modulates the stoichiometry of A3G in the complex. We applied single molecule high-speed AFM to directly visualize the dynamics of A3G in the complexes. We were able to visualize A3G sliding and protein association-dissociation events. During sliding, A3G translocated over a 69 nucleotide ssDNA segment in less than 1 second. Association-dissociation events were more complex, as dimeric A3G could dissociate from the template as a whole, or undergo a two-step process with monomers capable of sequential dissociation. We conclude that A3G monomers, dimers, and higher order oligomers can bind ssDNA substrates independent of strand polarity and availability of free ssDNA ends.
PMCID: PMC3448016  PMID: 22809226
APOBEC3G; single-stranded DNA binding proteins; site search mechanisms; atomic force microscopy; AFM; high speed-AFM
15.  Imaging of nucleic acids with atomic force microscopy 
Methods (San Diego, Calif.)  2011;54(2):274-283.
Atomic force microscopy (AFM) is a key tool of nanotechnology with great importance in applications to DNA nanotechnology and to the recently emerging field of RNA nanotechnology. Advances in the methodology of AFM now enable reliable and reproducible imaging of DNA of various structures, topologies, and DNA and RNA nanostructures. These advances are reviewed here with emphasis on methods utilizing modification of mica to prepare the surfaces enabling reliable and reproducible imaging of DNA and RNA nanostructures. Since the AFM technology for DNA is more mature, AFM imaging of DNA is introduced in this review to provide experience and background for the improvement of AFM imaging of RNA. Examples of imaging different structures of RNA and DNA are discussed and illustrated. Special attention is given to the potential use of AFM to image the dynamics of nucleic acids at the nanometer scale. As such, we review recent advances with the use of time-lapse AFM.
PMCID: PMC3114274  PMID: 21310240
Atomic force microscopy; AFM; DNA dynamics; DNA nanostructures; Holliday junctions; RNA assembly; RNA nanostructures; High-speed AFM
16.  Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein 
PLoS ONE  2012;7(5):e38099.
Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn.
PMCID: PMC3360652  PMID: 22662273
17.  The Role of Histone H4 Biotinylation in the Structure of Nucleosomes 
PLoS ONE  2011;6(1):e16299.
Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation.
Methodology/Principal Findings
To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p<0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA.
The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation.
PMCID: PMC3029316  PMID: 21298003
18.  Atomic Force Microscopy Studies Provide Direct Evidence for Dimerization of the HIV Restriction Factor APOBEC3G* 
The Journal of Biological Chemistry  2010;286(5):3387-3395.
APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.
PMCID: PMC3030345  PMID: 21123176
Atomic Force Microscopy; DNA-binding Protein; DNA-Protein Interaction; HIV; Protein-DNA Interaction; Single Molecule Biophysics
19.  Single Molecule Dynamics of the DNA-EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy 
Biochemistry  2009;48(44):10492-10498.
The study of protein interactions with DNA is important to gain a fundamental understanding of how numerous biological processes occur, including recombination, transcription, repair, etc. In this study, we use the EcoRII restriction enzyme, which employs a three-site binding mechanism in order to catalyze cleavage of a single recognition site. Using high-speed atomic force microscopy (HS-AFM) to image single-molecule interactions in real time, we were able to observe binding, translocation, and dissociation mechanisms of the EcoRII protein. The results show that the protein can translocate along DNA to search for the specific binding site. Also, once specifically bound at a single site, the protein is capable of translocating along the DNA to locate the second specific binding site. Furthermore, two alternative modes of dissociation of the EcoRII protein from the loop structure were observed, which result in the protein stably bound as monomers to two sites or bound to a single site as a dimer. From these observations, we propose a model in which this pathway is involved in the formation and dynamics of a catalytically active three-site complex.
PMCID: PMC2784205  PMID: 19788335
20.  DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme 
Nucleic Acids Research  2010;38(20):7142-7154.
To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer–dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.
PMCID: PMC2978343  PMID: 20571089
21.  Dynamic properties of pH-dependent structural organization of the amyloidogenic β-protein (1–40) 
Prion  2009;3(1):31-43.
The structural organization of the amyloidogenic β-protein containing 40 amino acid residues (Aβ40) was studied by the high temperature molecular dynamics simulations in the acidic (pH ∼ 3) and basic (pH ∼ 8) pH regions. The obtained data suggest that the central Ala21-Gly29 segment of Aβ40 can adopt folded and partially unfolded structures. At the basic pH, this segment forms folded structures stabilized by electrostatic interactions and hydrogen bonds. At the acidic pH, it forms partially unfolded structures. Two other segments flanking to the central segment exhibit the propensity to adopt unstable interconverting α-helical, 310-helical and turn-like structures. One of these segments is comprised of the Ala30-Val36 residues at both of the considered pHs. The second segment is comprised of the Glu11-Phe20 at the basic pH and of the Glu11-Val24 residues at the acidic pHs. The revealed pH-dependent structuration of the Aβ40 allowed us to suggest a possible scenario for initial Aβ aggregation. According to this scenario, the occurrence of the partially unfolded states of the Ala21-Gly29 segment plays main role in the Aβ oligomerization process.
PMCID: PMC2676741  PMID: 19372746
amyloid-β protein; Alzheimer disease; oligomerization; fibril; electrostatic interactions; molecular dynamics simulations
22.  Quantitative Analyses of RAG-RSS Interactions and Conformations Revealed by Atomic Force Microscopy† 
Biochemistry  2008;47(43):11204-11211.
During V(D)J recombination, the site specific DNA excision is dictated by the binding of RAG1/2 proteins to the conserved recombination signal sequence (RSS) within the genome. The interaction between RAG1/2 and RSS is thought to involve a large DNA distortion that is permissive for DNA cleavage. In this study, using atomic force microscopy imaging (AFM), we analyzed individual RAG-RSS complexes, in which the bending angle of RAG-associated RSS substrates could be visualized and quantified. We provided the quantitative measurement on the conformations of specific RAG-12RSS complexes. Previous data indicating the necessity of RAG2 for recombination implies a structural role in the RAG-RSS complex. Surprisingly however, no significant difference was observed in conformational bending with AFM between RAG1-12RSS and RAG1/2-12RSS. RAG1 was found sufficient to induce DNA bending and the addition of RAG2 did not change the bending profile. In addition, a pre-nicked 12RSS bound by RAG1/2 proteins displayed a conformation similar to the one observed with the intact 12RSS, implicating that no greater DNA bending occurs after the nicking step in the signal complex. Taken together, the quantitative AFM results on the components of the recombinase emphasize a tightly held complex with a bend angle value near 60°, which may be a prerequisite step for the site-specific nicking by the V(D)J recombinase.
PMCID: PMC2648828  PMID: 18831563
DNA; V(D)J Recombination; RAG1; RAG2; AFM; DNA bending
23.  Probing interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy 
Journal of molecular biology  2006;365(5):1407-1416.
SfiI belongs to a family of restriction enzymes that function as tetramers binding two recognition regions for the DNA cleavage reaction. SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site specific binding. SfiI enzymatic action has been very well characterized. However, properties of the complex prior to the cleavage reaction are not clear yet. We applied AFM single molecule force spectroscopy to analyze the strength of interactions within the SfiI - DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and another one to the AFM probe. The complex was formed by the protein present in the solution. An alternative setup in which the protein was anchored to the surface allowed us to probe the stability of the complex formed with one duplex only in the approach-retraction experiments, with the duplex immobilized at the AFM tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy - DFS). The DFS data suggest that the dissociation reaction of SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was also instrumental in revealing the role of the 5 base pair spacer region within the palindromic recognition site on DNA-SfiI complex stability. The data show that, although the change of nonspecific sequence does not alter the position of activation barrier, it significantly changes values of the off rates.
PMCID: PMC1847770  PMID: 17125791
synaptic complex; force spectroscopy; protein-DNA interactions; AFM; site-specific recognition
24.  The Effect of DNA Supercoiling on Geometry of Holliday Junctions† 
Biochemistry  2006;45(43):12998-13006.
Unusual DNA conformations including cruciforms play an important role in gene regulation and various DNA transactions. Cruciforms are also the models for Holliday junctions, the transient DNA conformations critically involved in DNA homologous and site-specific recombination, repair and replication. Although the conformations of immobile Holliday junctions in linear DNA molecules have been analyzed with use of various techniques, the role of DNA supercoiling has not been studied systematically. We utilized Atomic Force Microscopy (AFM) to visualize cruciform geometry in plasmid DNA with different superhelical densities at various ionic conditions. Both folded and unfolded conformations of the cruciform were identified, and the data showed that DNA supercoiling shifts the equilibrium between folded and unfolded conformations of the cruciform towards the folded one. In topoisomers with low superhelical density the population of folded conformation is 50 to 80 %, depending on ionic strength of the buffer and a type of cation added, whereas in the sample with high superhelical density, this population is as high as 98-100%. The time-lapse studies in aqueous solutions allowed us to observe the conformational transition of the cruciform directly. The time-dependent dynamics of the cruciform correlates with the structural changes revealed by the ensemble-averaged analysis of dry samples. Altogether, the data obtained show directly that DNA supercoiling is the major factor determining the Holliday junction conformation.
PMCID: PMC1646289  PMID: 17059216
25.  DNA Strand Arrangement within SfiI-DNA Complex: Atomic Force Microscopy Analysis† 
Biochemistry  2006;45(1):152-158.
The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before a complete cleavage of four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under non-cleaving reaction conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by the interaction with two binding sites in one DNA molecule (cis interaction) as well as the complexes obtained by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex the angles between the DNA strands at the complex proximity regardless of the complex type were systematically analyzed. All the data show a clear-cut bimodal distributions centered around peak values corresponding to 60° and 120°. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA templates with strands of different lengths and with different locations of the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles 60° or 120° between the strands corresponds to complex in which one of the strands flipped the orientation relative to another. Both types of complexes for 5 different sequences in the center are present almost equally. This finding suggests that there is no preferential orientation of the DNA cognate site within the complex suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein.
PMCID: PMC1352315  PMID: 16388590
DNA synaptic complexes; protein-DNA interaction; restriction-modification; DNA looping; scanning probe microscopy

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