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2.  Visualization of DNA and Protein-DNA Complexes with Atomic Force Microscopy 
This article describes sample preparation techniques for AFM imaging of DNA and protein–DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. 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. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purifi cation of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein–DNA complexes.
doi:10.1007/978-1-62703-776-1_17
PMCID: PMC4226758  PMID: 24357372
Atomic force microscopy; AFM; mica functionalization; surface chemistry; silanes; silatranes; DNA structure and dynamics; protein-DNA complexes
3.  Atomic Force Microscopy Studies of APOBEC3G Oligomerization and Dynamics 
Journal of structural biology  2013;184(2):10.1016/j.jsb.2013.09.008.
The DNA cytosine deaminase APOBEC3G (A3G) is a two-domain protein that binds single-stranded DNA (ssDNA) largely through its N-terminal domain and catalyzes deamination using its C-terminal domain. A3G is considered an innate immune effector protein, with a natural capacity to block the replication of retroviruses such as HIV and retrotransposons. However, knowledge about its biophysical properties and mechanism of interaction with DNA are still limited. Oligomerization is one of these unclear issues. What is the stoichiometry of the free protein? What are the factors defining the oligomeric state of the protein? How does the protein oligomerization change upon DNA binding? How stable are protein oligomers? We address these questions here using atomic force microscopy (AFM) to directly image A3G protein in a free-state and in complexes with DNA, and using time-lapse AFM imaging to characterize the dynamics of A3G oligomers. We found that the formation of oligomers is an inherent property of A3G and that the yield of oligomers depends on the protein concentration. Oligomerization of A3G in complexes with ssDNA follows a similar pattern: the higher the protein concentrations the larger oligomers sizes. The specificity of A3G binding to ssDNA does not depend on stoichiometry. The binding of large A3G oligomers requires a longer ssDNA substrate; therefore, much smaller oligomers form complexes with short ssDNA. A3G oligomers dissociate spontaneously into monomers and this process primarily occurs through a monomer dissociation pathway.
doi:10.1016/j.jsb.2013.09.008
PMCID: PMC3844295  PMID: 24055458
APOBEC3G; single-stranded DNA binding proteins; site search mechanisms; atomic force microscopy; AFM; high-speed AFM
4.  Alpha-synuclein misfolding assessed with single molecule AFM force spectroscopy: Effect of pathogenic mutations 
Biochemistry  2013;52(42):10.1021/bi401037z.
Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson’s disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early-onset PD; however the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central non-amyloid-beta component (NAC) of the protein, but rather expand beyond this segment. All three mutations alter the protein’s folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed.
doi:10.1021/bi401037z
PMCID: PMC3844294  PMID: 24066883
5.  Novel Polymer Linkers for Single Molecule AFM Force Spectroscopy 
Methods (San Diego, Calif.)  2013;60(2):161-168.
Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aβ42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications.
doi:10.1016/j.ymeth.2013.02.019
PMCID: PMC3698867  PMID: 23624104
6.  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+.
doi:10.1007/s11481-012-9416-6
PMCID: PMC3586772  PMID: 23143330
Amyloid β-protein; Aβ42; Alzheimer’s disease; Cu2+ cations; Single molecule force spectroscopy; Atomic force microscopy imaging
7.  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.
doi:10.1021/bi300733d
PMCID: PMC3448016  PMID: 22809226
APOBEC3G; single-stranded DNA binding proteins; site search mechanisms; atomic force microscopy; AFM; high speed-AFM
8.  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.
doi:10.1088/0953-8984/24/16/164205
PMCID: PMC3482402  PMID: 22466073
AFM; Force spectroscopy; protein aggregation; electrostatics; nanoimaging
9.  Nanoimaging methods for biomedicine 
Methods (San Diego, Calif.)  2013;60(2):111-112.
doi:10.1016/j.ymeth.2013.05.009
PMCID: PMC4227392  PMID: 23721609
10.  Fibrillogenesis of Huntingtin and Other Glutamine Containing Proteins 
Sub-cellular biochemistry  2012;65:225-251.
This chapter focuses on the aggregation of glutamine containing peptides and proteins with an emphasis on huntingtin protein, whose aggregation leads to the development of Huntington’s disease. The kinetics that leads to the formation of amyloids, the structure of aggregates of various types and the morphological mechanical properties of amyloid fibrils are described. The kinetics of amyloid fibril formation has been proposed to follow a nucleation dependent polymerization model, dependent upon the size of the nucleus. This model and the effect of the polyglutamine length on the nucleus size are reviewed. Aggregate structure is characterized at two different levels. The atomic-scale resolution structure of fibrillar and crystalline aggregates of polyglutamine containing proteins and peptides was determined by X-ray crystallography and solid-state nuclear magnetic resonance (NMR). The chapter outlines the results obtained by both these techniques. Atomic force microscopy (AFM) was instrumental in elucidating the morphology of fibrils, their organization and assembly. The chapter also discusses the high stability of amyloid fibrils, including their mechanical properties as revealed by AFM.
doi:10.1007/978-94-007-5416-4_10
PMCID: PMC4226413  PMID: 23225006
Huntington’s disease; Amyloids; Self-assembly; Fibrillogenesis; Atomic Force Microscopy; AFM; Solid-state NMR
11.  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.
doi:10.1016/j.micron.2010.08.011
PMCID: PMC2997872  PMID: 20864349
AFM; surface functionalization; silanes; DNA; DNA supercoiling; alternative DNA structures; protein-DNA complexes; single molecule imaging
12.  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.
doi:10.1155/2012/650840
PMCID: PMC3268019  PMID: 22312477
13.  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.
doi:10.4161/pri.4.4.13125
PMCID: PMC3268959  PMID: 20724837
protein misfolding; prion; atomic force microscopy; nanomedicine; force spectroscopy
14.  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
15.  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.
doi:10.1021/bi900977t
PMCID: PMC2752652  PMID: 19618963
16.  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.
doi:10.1016/j.ymeth.2008.09.002
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
17.  Interaction of APOBEC3A with DNA Assessed by Atomic Force Microscopy 
PLoS ONE  2014;9(6):e99354.
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.
doi:10.1371/journal.pone.0099354
PMCID: PMC4048275  PMID: 24905100
18.  Molecular Mechanism of Misfolding and Aggregation of Aβ (13–23) 
The journal of physical chemistry. B  2013;117(20):6175-6186.
The misfolding and self-assembly of the amyloid-beta (Aβ) peptide into aggregates is a molecular signature of the development of Alzheimer’s disease but molecular mechanisms of the peptide aggregation remain unknown. Here, we combined Atomic Force Microscopy (AFM) and Molecular Dynamics (MD) simulations to characterize misfolding process of an Aβ peptide. Dynamic force spectroscopy AFM analysis showed that the peptide forms stable dimers with the lifetime of ~1 s. During MD simulations isolated monomers gradually adopt essentially similar non-structured conformations independent from the initial structure. However, when two monomers approach their structure changes dramatically and the conformational space for the two monomers become restricted. The arrangement of monomers in antiparallel orientation leads to the cooperative formation of β-sheet conformation. Interactions, including hydrogen bonds, salt bridges and weakly polar interactions of side chains stabilize the structure of the dimer. Under the applied force, the dimer, as during the AFM experiments, dissociates in a cooperative manner. Thus, misfolding of the Aβ peptide proceeds via the loss of conformational flexibility and formation of stable dimers suggesting their key role in the subsequent Aβ aggregation process.
doi:10.1021/jp402938p
PMCID: PMC3695694  PMID: 23642026
Amyloid beta; aggregation; atomic force microscopy; molecular dynamics simulations; stable dimers
19.  Centromere chromatin: a loose grip on the nucleosome? 
doi:10.1038/nsmb.2745
PMCID: PMC3970403  PMID: 24389544
20.  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.
doi:10.1021/bi301074r
PMCID: PMC3580866  PMID: 23286197
MIF4GD; translation initiation; oligomerization; intrinsically disordered protein; phosphorylation; histone; SLBP; SLIP1
21.  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.
doi:10.1007/978-1-62703-056-4_14
PMCID: PMC3848609  PMID: 23027008
Atomic force microscopy; AFM; Mica functionalization; Surface chemistry; Silanes; Silatranes; DNA structure and dynamics; Protein-DNA complexes
22.  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.
doi:10.1021/bi201863z
PMCID: PMC3848610  PMID: 22304461
23.  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.
doi:10.1038/srep02880
PMCID: PMC3791449  PMID: 24096987
24.  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.
doi:10.1016/j.ymeth.2011.02.001
PMCID: PMC3114274  PMID: 21310240
Atomic force microscopy; AFM; DNA dynamics; DNA nanostructures; Holliday junctions; RNA assembly; RNA nanostructures; High-speed AFM
25.  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.
doi:10.1371/journal.pone.0038099
PMCID: PMC3360652  PMID: 22662273

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