Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.     

Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy

Filamin A (ABP-280), which is an actin-binding protein of 560 kDa as a dimer, can, together with actin filaments, produce an isotropic cross-linked three-dimensional network (actin/filamin A gel) that plays an important role in mechanical responses of cells in processes such as maintenance of membrane stability and translational locomotion. In this study, we investigated the mechanical properties of single filamin A molecules using atomic force microscopy. In force-extension curves, we observed sawtooth patterns corresponding to the unfolding of individual immunoglobulin (Ig)-fold domains of filamin A. At a pulling speed of 0.37 microm/s, the unfolding interval was sharply distributed around 30 nm, while the unfolding force ranged from 50 to 220 pN. This wide distribution of the unfolding force can be explained by variation in values of activation energy and the width of activation barrier of 24 Ig-fold domains of the filamin A at the unfolding transition. This unfolding can endow filamin A with great extensibility. The refolding of the unfolded chain of filamin A occurred when the force applied to the protein was reduced to near zero, indicating that its unfolding is reversible. Based on these results, we discuss here the physiological implications of the mechanical properties of single filamin A molecules.     

Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.     

Macromolecular crystal growth as revealed by atomic force microscopy

Direct visualization of macromolecular crystal growth using atomic force microscopy (AFM) has provided a powerful tool in the delineation of mechanisms and the kinetics of the growth process. It has further allowed us to evaluate the wide variety of impurities that are incorporated into crystals of proteins, nucleic acids, and viruses. We can, using AFM, image the defects and imperfections that afflict these crystals, the impurity layers that poison their surfaces, and the consequences of various factors on morphological development. All of these can be recorded under normal growth conditions, in native mother liquors, over time intervals ranging from minutes to days, and at the molecular level.     

Nanoscopic lipid domain dynamics revealed by atomic force microscopy

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.     

Mechanical unfolding pathways of the enhanced yellow fluorescent protein revealed by single molecule force spectroscopy

We used single molecule force spectroscopy to characterize the mechanical stability of the enhanced yellow fluorescent protein (EYFP) (a mutant form of the green fluorescent protein (GFP)) and two of its circularly permutated variants. In all three constructs, we found two main unfolding peaks; the first corresponds to a transition state placed close to the termini and the second to a transition state placed halfway through the molecule. We attribute the second transition state to the shear rupture of the beta1- and beta6-strands, which we verified by introducing a point mutation in this region. Although both unfolding peaks were observed in all three EYFP variants, their relative frequency of occurrence varied. Our results demonstrated that the mechanical unfolding pathways in EYFP could be deciphered through the use of circular permutation.     

Surface ultrastructure of SARS coronavirus revealed by atomic force microscopy

Atomic force microscopy has been used to probe the surface nanostructures of severe acute respiratory syndrome coronavirus (SARS-CoV). Single crown-like virion was directly visualized and quantitative measurements of the dimensions for the structural proteins were provided. A corona of large, distinctive spikes in the envelope was measured after treatment with hydroxyoctanoic acid. High-resolution images revealed that the surface of each single SARS-CoV was surrounded with at least 15 spherical spikes having a diameter of 7.29 +/- 0.73 nm, which is in close agreement with that of S glycoproteins earlier predicted through the genomes of SARS-CoV. This study represents the first direct characterization of the surface ultrastructures of SARS-CoV particles at the nanometre scale and offers new prospects for mapping viral surface properties.     

Sequential assembly of collagen revealed by atomic force microscopy.

Most polymers which comprise biological filaments assemble by two mechanisms: nucleation and elongation or a sequential, stepwise process involving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filament. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correlated with turbidimetric alterations in the assembly mixture. The assembly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of assembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments approximately 2-6 nm in diameter (> 10 microns in length) many with a conspicuous approximately 67-nm axial period were observed. Occasional mature collagen fibrils with a approximately 67-nm axial repeat were found late in the course of assembly. Our results are consistent with initial end-to-end axial association of monomers to form oligomers followed by lateral association into higher-order filaments. On this basis, there appears to be at least two distinctive types of structural interactions (axial and lateral) which are operative at different levels in the assembly hierarchy of collagen.     

Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy,protein engineering and molecular dynamics simulations

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.     

Sequential unfolding of ankyrin repeats in tumor suppressor p16

The ANK repeat is a ubiquitous 33-residue motif that adopts a beta hairpin helix-loop-helix fold. Multiple tandem repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly by short-range interactions between residues close in sequence. The tumor suppressor p16(INK4) consists of four repeats and represents the minimal ANK folding unit. We found from Phi value analysis that p16 unfolded sequentially. The two N-terminal ANK repeats, which are distorted from the canonical ANK structure in all INK4 proteins and which are important for functional specificity, were mainly unstructured in the rate-limiting transition state for folding/unfolding, while the two C-terminal repeats were fully formed. A sequential unfolding mechanism could have implications for the cellular fate of wild-type and cancer-associated mutant p16 proteins.     

Unexpected binding motifs for subnucleosomal particles revealed by atomic force microscopy

The structure of individual nucleosomes organized within reconstituted 208-12 arrays at different levels of compaction was examined by tapping mode atomic force microscopy in air and liquid. Reconstitution at lower histone octamer to DNA weight ratios showed an extended beads-on-a-string morphology with less than the expected maximum of 12 nucleosome core particles per array, each particle located in the most favored positioning site. A correlation of the contour lengths of these arrays with the number of observed particles revealed two distinct populations of particles, one with approximately 50 nm of bound DNA and a second population with approximately 25 nm. The measured nucleosome center-to-center distances indicate that this approximately 25 nm is not necessarily symmetrically bound about the dyad axis, but can also correspond to DNA bound from either the entry or exit point of the particle to a location at or close to the dyad axis. An assessment of particle heights suggests that particles wrapping approximately 25 nm of DNA are most likely to be subnucleosomal particles, which lack either one or both H2A-H2B dimers. At a higher reconstitution ratio, folded compact arrays fully populated with 12 nucleosome core particles, were observed. Liquid measurements demonstrated dynamic movements of DNA loops protruding from these folded arrays.     

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.     

Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy

During V(D)J recombination, 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 prenicked 12RSS bound by RAG1/2 proteins displayed a conformation similar to the one observed with the intact 12RSS, implying 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 degrees , which may be a prerequisite step for the site-specific nicking by the V(D)J recombinase.     

Study of lamellae of a recombinant spider-web protein by atomic force microscopy

Lamellae formed on mica by protein 1F9, a recombinant analog of spider web protein, have been studied by atomic force microscopy. It is shown that the molecules of 1F9 dissolved in strong solvents are capable of aggregating on the mica surface to form lamellae less than 1 nm in height and more than 1 μm in length. A model of a plane zigzag has been constructed to describe the conformation of 1F9 molecules on the mica surface.     

Imaging of single hairpin ribozymes in solution by atomic force microscopy.

The hairpin ribozyme is a short endonucleolytic RNA motif isolated from a family of related plant virus satellite RNAs. It consists of two independently folding domains, each comprising two Watson-Crick helices flanking a conserved internal loop. The domains need to physically interact (dock) for catalysis of site-specific cleavage and ligation reactions. Using tapping-mode atomic force microscopy in aqueous buffer solution, we were able to produce high quality images of individual hairpin ribozyme molecules with extended terminal helices. Three RNA constructs with either the essential cleavage site guanosine or a detrimental adenosine substitution and with or without a 6-nt insertion to confer flexibility to the interdomain hinge show structural differences that correlate with their ability to form the active docked conformation. The observed contour lengths and shapes are consistent with previous bulk-solution measurements of the transient electric dichroism decays for the same RNA constructs. The active docked construct appears as an asymmetrically docked conformation that might be an indication of a more complicated docking event than a simple collapse around the interdomain hinge.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy

A small container of several to a few hundred µm³ (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of ~80 nm was found. In addition to this ‘80 nm fiber’, a thinner ‘40 nm fiber’ and a higher order ‘loop’ structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a ‘coral reef structure’ that also possessed the 80 nm fiber units, and, finally, into a ‘tightly compacted nucleoid’ that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid.     

New internal structure of spider dragline silk revealed by atomic force microscopy.

Atomic force microscopy was used to study the three-dimensional nanometer-scale structure of the dragline silk of Nephila clavipes from microtomed sections of the silk. Contrary to a previously proposed model of randomly distributed protein crystallites interspersed in amorphous regions, a highly organized skin-core structure of the fiber was observed. The skin appeared to be thin with no discernible distinct features. The core consists of pleated fibril-like structures, which are arranged in two concentric cylinders. Upon stretching, the pleats were smoothed out substantially. The mechanical properties of spider silk can quite straightforwardly be related to the newly observed structures.