High-resolution visualization of fibrinogen molecules and fibrin fibers with atomic force microscopy

We report an atomic force microscopy (AFM) study of fibrinogen molecules and fibrin fibers with resolution previously achieved only in few electron microscopy images. Not only are all objects triads, but the peripheral D regions are resolved into the two subdomains, apparently corresponding to the βC and γC domains. The conformational analysis of a large population of fibrinogen molecules on mica revealed the two most energetically favorable conformations characterized by bending angles of ～100 and 160 degrees. Computer modeling of the experimental images of fibrinogen molecules showed that the AFM patterns are in good agreement with the molecular dimensions and shapes detected by other methods. Imaging in different environments supports the expected hydration of the fibrinogen molecules in buffer, whereas imaging in humid air suggests the 2D spreading of fibrinogen on mica induced by an adsorbed water layer. Visualization of intact hydrated fibrin fibers showed cross-striations with an axial period of 24.0 ± 1.6 nm, in agreement with a pattern detected earlier with electron microscopy and small-angle X-ray diffraction. However, this order is clearly detected on the surface of thin fibers and becomes less discernible with the fiber's growth. This structural change is consistent with the proposal that thinner fibers are denser than thicker ones, that is, that the molecule packing decreases with the increasing of the fibers' diameter.     

A convenient method of aligning large DNA molecules on bare mica surfaces for atomic force microscopy.

Large DNA molecules remain difficult to be imaged by atomic force microscopy (AFM) because of the tendency of aggregation. A method is described to align long DNA fibers in a single direction on unmodified mica to facilitate AFM studies. The clear background, minimal overstretching, high reproducibility and convenience of this aligning procedure make it useful for physical mapping of genome regions and the studies of DNA-protein complexes.     

Detecting ultraviolet damage in single DNA molecules by atomic force microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.     

A new, simple method for linking of antibodies to atomic force microscopy tips

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.     

Cationic peptide-induced remodelling of model membranes: direct visualization by in situ atomic force microscopy

Our understanding of how antimicrobial and cell-penetrating peptides exert their action at cell membranes would benefit greatly from direct visualization of their modes of action and possible targets within the cell membrane. We previously described how the cationic antimicrobial peptide, indolicidin, interacted with mixed zwitterionic planar lipid bilayers as a function of both peptide concentration and lipid composition [Shaw, J.E. et al., 2006. J. Struct. Biol. 154 (1), 42-58]. In the present report, in situ atomic force microscopy was used to characterize the interactions between three families of cationic peptides: (1) tryptophan-rich antimicrobial peptides--indolicidin and two of its analogues, (2) an amphiphilic alpha-helical membranolytic peptide--melittin, and (3) an arginine-rich cell-penetrating peptide--Tat with phase-separated planar bilayers containing 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) or DOPC/N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM)/cholesterol. We found that these cationic peptides all induced remodelling of the model membranes in a concentration, and family-dependent manner. At low peptide concentration, these cationic peptides, despite their different biological roles, all appeared to reduce the interfacial line tension at the domain boundary between the liquid-ordered and liquid-disordered domains. Only at high peptide concentration was the membrane remodelling induced by these peptides morphologically distinct among the three families. While the transformation caused by indolicidin and its analogues were structurally similar, the concentration required to initiate the transformation was strongly dependent on the hydrophobicity of the peptide. Our use of lipid compositions with no net charge minimized the electrostatic interactions between the cationic peptides and the model supported bilayers. These results suggest that peptides within the same functional family have a common mechanism of action, and that membrane insertion of short cationic peptides at low peptide concentration may also alter membrane structure through a common mechanism regardless of the peptide's origin.     

Viewing molecules with scanning tunneling microscopy and atomic force microscopy

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.     

Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.     

High-resolution atomic force microscopy of DNA

A method using high resolution atomic force microscopy for imaging DNA has been elaborated. Using super-sharp probes and modified graphite as support for molecule adsorption, DNA molecule images were obtained whose resolution made possible the observation of their fine structure with repeated helical motifs. The method can be used to visualize individual spread molecules of single-stranded DNA.     

Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy

Attempts were made to apply atomic force microscopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules. In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing. The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA. Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin–avidin complex. Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites. This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample.     

High-resolution atomic force microscopy visualization of metalloproteins and their complexes

Background

Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level.

Structure of branched DNA molecules: gel retardation and atomic force microscopy studies.

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.     

Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.     

Atomic force microscopy of DNA molecules.

DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Graphical method for force analysis: macromolecular mechanics with atomic force microscopy

We present a graphical method for a unifying, quantitative analysis of molecular bonding-force measurements by atomic force microscopy (AFM). The method is applied to interpreting a range of phenomena commonly observed in the experimental AFM measurements of noncovalent, weak bonds between biological macromolecules. The analysis suggests an energy landscape underlying the intermolecular force and demonstrates that many observations, such as "snaps-on," "jumps-off," and hysteresis loops, are different manifestations of a double-well energy landscape. The analysis gives concrete definitions for the operationally defined "attractive" and "adhesive" forces in terms of molecular parameters. It is shown that these operationally defined quantities are usually functions of the experimental setup, such as the stiffness of the force probe and the rate of its movement. The analysis reveals a mechanical instability due to the multistate nature of molecular interactions and provides new insight into macromolecular viscosity. The graphical method can equally be applied to a quantitative analysis of multiple unfolding of subunits of the giant muscle protein titin under AFM.     

Rapid self-assembly of alpha-synuclein observed by in situ atomic force microscopy

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.     

In situ imaging of detergent-resistant membranes by atomic force microscopy

Purified detergent-resistant membranes (DRMs) are powerful tools for the biochemical study of plasma membrane domains. To what extent these isolated DRMs correspond to native membrane domains remains, however, a matter of debate. The most immediate question to be answered concerns the in situ size range of DRMs, a determination that escapes classical microscopy techniques. In this study we show that in situ three-dimensional images of a material as fragile as Triton X-100-treated cells can be obtained, in buffer, by tapping mode atomic force microscopy. These images establish that, prior to the isolation procedure, the detergent plasma membrane fragments form domains whose size frequently exceeds 15-20 microm(2). This DRMs size range is about 1 order of magnitude higher than that estimated for the larger microdomains of living cells, which strongly suggests that membrane microdomains rearrange into larger DRMs during Triton X-100 treatment. Concomitantly, the images also reveal the presence of the cytoskeleton, which is resistant to detergent extraction, and suggest that, in situ, DRMs are associated with the membrane cytoskeleton.