alpha-Synuclein misfolding: single molecule AFM force spectroscopy study

Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson’s disease, induced by misfolding of α-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of α-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized α-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected α-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded α-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, α-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of α-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of α-synuclein.     

Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements

Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K(ds)) to double-stranded DNA to determine K(ds) as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K(ss), the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD.     

Mechanical design of the first proximal Ig domain of human cardiac titin revealed by single molecule force spectroscopy

The elastic I-band part of muscle protein titin contains two tandem immunoglobulin (Ig) domain regions of distinct mechanical properties. Until recently, the only known structure was that of the I27 module of the distal region, whose mechanical properties have been reported in detail. Recently, the structure of the first proximal domain, I1, has been resolved at 2.1A. In addition to the characteristic beta-sandwich structure of all titin Ig domains, the crystal structure of I1 showed an internal disulfide bridge that was proposed to modulate its mechanical extensibility in vivo. Here, we use single molecule force spectroscopy and protein engineering to examine the mechanical architecture of this domain. In contrast to the predictions made from the X-ray crystal structure, we find that the formation of a disulfide bridge in I1 is a relatively rare event in solution, even under oxidative conditions. Furthermore, our studies of the mechanical stability of I1 modules engineered with point mutations reveal significant differences between the mechanical unfolding of the I1 and I27 modules. Our study illustrates the varying mechanical architectures of the titin Ig modules.     

Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.     

Measurement of membrane binding between recoverin,a calcium-myristoyl switch protein,and lipid bilayers by AFM-based force spectroscopy

Myristoyl switch is a feature of several peripheral membrane proteins involved in signal transduction pathways. This unique molecular property is best illustrated by the "Ca(2+)-myristoyl switch" of recoverin, which is a Ca(2+)-binding protein present in retinal rod cells of vertebrates. In this transduction pathway, the Ca(2+)-myristoyl switch acts as a calcium sensor involved in cell recovery from photoactivation. Ca(2+) binding by recoverin induces the extrusion of its myristoyl group to the solvent, which leads to its translocation from cytosol to rod disk membranes. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the extent of membrane binding of recoverin in the absence and presence of calcium, and to quantify this force of binding. An adhesion force of 48 +/- 5 pN was measured between recoverin and supported phospholipid bilayers in the presence of Ca(2+). However, no binding was observed in the absence of Ca(2+). Experiments with nonmyristoylated recoverin confirmed these observations. Our results are consistent with previously measured extraction forces of lipids from membranes.     

Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k_off, compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.     

Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: preparation, bulk and single molecule spectroscopy

Reconstitution of the 16 kDa N-terminal domain of the peridinin-chlorophyll-protein, N-PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N-PCP. Besides homo-chlorophyllous complexes, hetero-chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo- and hetero-chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.     

Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy

p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.     

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores.     

Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications

Recent developments in single molecule force spectroscopy (SMFS) allow direct observation and measurements of forces that hold protein-DNA complexes together. Furthermore, the mechanics of double-stranded (ds) DNA molecules in the presence of small binding ligands can be detected. The results elucidate molecular binding mechanisms and open the way for ultra sensitive and powerful biosensor applications.     

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF₁₆₅ was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF₁₆₅ was dependent on the concentration of VEGF₁₆₅. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF₁₆₅ complexes was revealed, which was similar to that of the intact aptamer/VEGF₁₆₅. Besides, the dissociation rate constant (k_off) of split aptamer/VEGF₁₆₅ was close to that of intact aptamer/VEGF₁₆₅, and the interaction force of split aptamer/VEGF₁₆₅ was higher than the force of intact aptamer/VEGF₁₆₅. It indicated that split aptamer also possessed high affinity with VEGF₁₆₅. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level.     

Specific molecular interactions by force spectroscopy: from single bonds to collective properties

Interactions involving multiple bonds occur throughout biology, and have distinct properties that are fundamentally different from those present in single bond systems. We have developed a new method to analyse the AFM force measurements in order to extract relevant information and to characterise the interactions involving from single to multiple bonds. Our study reveals a surprising behaviour in the presence of multiple bonds with a high rebinding probability: the mean binding forces increase with decreasing pulling velocity. Such behaviour is different from the force dependence on the loading rate for single bond rupture or existing models for multiple bonds rupture.     

Understanding the elasticity of fibronectin fibrils: unfolding strengths of FN-III and GFP domains measured by single molecule force spectroscopy.

While it is well established that fibronectin (FN) matrix fibrils are elastic, the mechanism of fibril elasticity during extension is still debated. To investigate the molecular origin of FN fibril elasticity, we used single molecule force spectroscopy (SMFS) to determine the unfolding behavior of a recombinant FN-III protein construct that contained eight FN-III domains ((1-8)FN-III) and two green fluorescent protein (GFP) domains. FN-III domains were distinguished from GFP domains by their shorter unfolding lengths. The unfolding strengths of both domains were determined for a wide range of pulling rates (50 to 1,745 nm/s). We found that the mechanical stabilities of FN-III and GFP domains were very similar to each other over the entire range of pulling speeds. FN fibrils containing GFP remain brightly fluorescent, even when stretched, meaning that GFP domains remain largely folded. Since GFP and FN-III have equal unfolding strengths, this suggests that FN-III domains are not extensively unraveled in stretched FN fibrils. Our results thus favor an alternative model, which invokes a conformational change from a compact to an extended conformation, as the basis for FN fibril elasticity.     

Ferritin: design and formation of an iron-storage molecule

Although essential for most forms of life, too much iron is harmful. To cope with these antagonistic phenomena an iron-storage molecule, ferritin, has evolved. The structure of horse spleen apoferritin, which has recently been refined, consists of 24 symmetrically related subunits forming a near-spherical hollow shell. In ferritin the central cavity is occupied by an iron core of 'ferrihydrite', a geologically ephemeral mineral found in hot or cold springs and in mine workings, or produced in the laboratory by heating solutions of ferric salts. Ferritin itself forms most readily from apoferritin, in the presence of dioxygen, from FeII, not FeIII. Access to its interior is through small intersubunit channels, and the protein influences both the rate of FeII-oxidation and the form of oxide produced.