Mechanical unfolding studies of protein molecules

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Probing Single Individual Proteins Unfold and Refold with 1-μs Resolution: Improved AFM-Based Single Molecule Force Spectroscopy

The study on the nanoparticle-polymer interactions is very important for the design/preparation of high performance polymer nanocomposite. Here we present a method to quantify the polymer-particle interaction at single molecule level by using AFM-based single molecule force spectroscopy (SMFS). As a proof-of-concept study, we choose poly-l-lysine (PLL) as the polymer and several different types of polyoxometalates (POM) as the model particles to construct several different polymer nanocomposites and to reveal the binding mode and quantify the binding strength in these systems. Our results reveal that the shape of the nanoparticle and the binding geometry in the composite have significantly influenced the binding strength of the PLL/POM complexes. Our dynamic force spectroscopy studies indicate that the disk-like geometry facilitate the unbinding of PLL/AlMo6 complexes in shearing mode, while the unzipping mode becomes dominate in spherical PLL-P8W48 system. We have also systematically investigated the effects of charge numbers, particle size, and ionic strength on the binding strength and binding mode of PLL/POM, respectively. Our results show that electrostatic interactions dominate the stability of PLL/POM complexes. These findings provide a way for tuning the mechanical properties of polyelectrolyte-nanoparticle composites.

Amorphous red phosphorus (ARP) has recently been identified as a zig-zag ladder polymer, yet its function beyond a low-cost phosphorus source remains unexplored. Here we report the first host-guest chemistry of ARP. In water, hydrophobic ARP threads through α- or β-cyclodextrin (CD) to produce water-soluble pseudo-polyrotaxanes, as evidenced by nuclear magnetic resonance (NMR) spectroscopy, atomic force microscopy (AFM) imaging and AFM-based single-molecule force spectroscopy (SMFS). β-CD, whose cavity diameter (0.60 nm) matches the 0.582 nm van-der-Waals width of ARP, assembles readily with ARP; α-CD, which has a smaller cavity, requires conformational distortion before assembly, producing only ∼30% fully threaded chains even after 10 days. Threading converts collapsed ARP globules into extended chains whose mechanical stiffness increases with CD coverage. After assembly with β-CD, the water solubility of ARP can be significantly enhanced, which builds a method to tune the hydrophilicity of an inorganic polymer chain without the need for traditional chemical modification. This work establishes ARP as an inorganic building block for supramolecular assembly, opening routes to phosphorus-based functional materials.

Atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) is one of the most useful methods in the investigation of intra- or intermolecular interactions. To simplify the sample system and the data analysis, real biological and material systems have usually been simplified, and the target molecule of interest is normally extracted and bridged between the AFM tip and substrate for study, which is an effective way of understanding the real systems. With the development of technology (including the improvement of sample immobilization method), it is possible now to directly investigate molecular interactions in living system and real materials. The information obtained will be more useful for the better control of relevant biological processes and the design of high performance polymer materials. In this paper, recent progresses in AFM-SMFS study of molecular interactions in living cells and polymer materials are reviewed.

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

Force and function: probing proteins with AFM-based force spectroscopy

Muscle elasticity strongly relies on the mechanical anchoring of the giant protein titin to both the sarcomere M-band and the Z-disk. Such strong attachment ensures the reversible dynamics of the stretching-relaxing cycles determining the muscle passive elasticity. Similarly, the design of biomaterials with enhanced elastic function requires experimental strategies able to secure the constituent molecules to avoid mechanical failure. Here we show that an engineered titin-mimicking protein is able to spontaneously dimerize in solution. Our observations reveal that the titin Z1Z2 domains are key to induce dimerization over a long-range distance in proteins that would otherwise remain in their monomeric form. Using single molecule force spectroscopy, we measure the threshold force that triggers the noncovalent transition from protein dimer to monomer, occurring at ∼700 piconewtons. Such extremely high mechanical stability is likely to be a natural protective mechanism that guarantees muscle integrity. We propose a simple molecular model to understand the force-induced dimer-to-monomer transition based on the geometric distribution of forces occurring within a dimeric protein under mechanical tension.

Molecular motors are ubiquitous in nature and are essential in controlling and performing numerous biological functions. They are able to rectify random motion to generate directional force and carry out macroscopic tasks (Schliwa in Molecular motors. Wiley-VCH, Weinheim, 2003 [1]). This ability has inspired attempts to create synthetic machines exhibiting control over rotary or linear motion (Kinbara and Aida in Chem Rev 105:1377–1400, 2005 [2], Kay et al. in Angew Chem Int Ed 46:72–191, 2007 [3]). Whereas some examples of synthetic systems able to use biased Brownian motion to perform work and collectively induce movement of much larger objects have been reported (Kay et al. in Angew Chem Int Ed 46:72–191, 2007 [3], Browne and Feringa in Nat Nanotech 1:25–35, 2006 [4], Berná et al. in Nat Mater 4:704–710, 2005 [5]), seeing such a single molecule at work remains a major challenge. Some elegant experiments on single-molecule machines adsorbed on a surface, imaged and manipulated with the tip of a scanning tunnelling microscope have been realized (Grill et al. in Nat Nanotech 2:95–98, 2007 [6], Manzano et al. in Nat Mater 8:576–579, 2009 [7]). However, in this case, the molecule is adsorbed on the surface, and the experiments are done in ultra-high vacuum at low temperature, which can be a limitation for systems designed to perform work in solution at room temperature or even in physiological conditions. Here, we discuss how a single synthetic small machine at work can be addressed by AFM-based single-molecule force spectroscopy, a tool able to monitor mechanical forces with sub-nanometer resolution and which has been widely used to investigate molecular-level processes in macromolecules and biological machines (Bustamante et al. in Annu Rev Biochem 73:705–748, 2004 [8], Special Issue in Annu Rev Biochem 77:45–228, 2008 [9], Evans in Annu Rev Biophys Biomol Struct 30:105–128, 2001 [10], Liang and Fernández in ACS Nano 3:1628–1645, 2009 [11], Puchner and Gaub in Curr Opin Struct Biol 19:605–614, 2009 [12]).KeywordsAtomic Force MicroscopyForce SpectroscopyThioctic AcidFluctuation TheoremAtomic Force Microscopy ForceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Pulling Geometry-Induced Errors in Single Molecule Force Spectroscopy Measurements

In this article, interactions between Bacillus subtilis single-stranded DNA binding proteins (BsSSB) and single-stranded DNA (ssDNA) were systematically studied. The effect of different molar ratios between BsSSB and ssDNA on their binding modes was first investigated by electrophoretic mobility shift assays (EMSAs). It is found that a high molar ratio of BsSSB to ssDNA can produce BsSSB-ssDNA complexes formed in the mode of two proteins binding one 65-nt (nucleotide) ssDNA whereas a low molar ratio facilitates the formation of BsSSB-ssDNA complexes in the mode of one protein binding one 65-nt ssDNA. Furthermore, two binding modes are in dynamic equilibrium. The unbinding force of BsSSB-ssDNA complexes was measured quantitatively in solutions with different salt concentrations by using AFM-based single-molecule force spectroscopy (SMFS). Our results show that the unbinding force is about 10 pN higher at high salt concentration (0.5 M NaCl) than at low salt concentration (0.1 M NaCl) and the lifetime of BsSSB-ssDNA complexes at high salt concentration is twice as long as that at low salt concentration. These results indicate that more tightly packed BsSSB-ssDNA complexes can form at high salt (0.5 M NaCl) concentration. In addition, the results of EMSA show that ssDNA, which is bound to BsSSB, can dissociate from BsSSB in the presence of the cDNA strand, indicating the dynamic nature of BsSSB-ssDNA interactions.

AFM-based single-molecule force spectroscopy (SMFS) is a powerful tool for the investigation of the elastic properties of a single polymer. Two typical organometallic polymers bearing ferrocene (Fc) groups in the backbone, poly(ferrocenyldimethylsilane) and poly(ferrocenylmethylphenylsilane), were investigated by SMFS to reveal their single-chain mechanical properties in normal and oxidized forms. We have found that the two polymers show similar elasticity in normal forms, though bearing different side groups. However, they exhibit different enthalpic elasticity after oxidation probably because of steric effects. Moreover, all experiments confirm that the single-chain elongation of poly(ferrocenyldimethylsilane) or poly(ferrocenylmethylphenylsilane) is reversible.

We report here a systematic investigation of the interactions between two heteropolymer DNA single-strands (ssDNA) and graphite surfaces using AFM-based single-molecule force spectroscopy (SMFS). For this purpose, force-displacement (FD) curves are recorded by peeling single-molecule ssDNA from graphite. We find that the unbinding forces are affected both by the DNA sequences and the ionic strength of the liquid environment. In particular, the unbinding force decreases with the increase of ionic strength. Dynamic force measurements indicate that the unbinding force increases nonlinearly with the logarithm of the applied loading rate. The force data at different loading rates can be fitted with a recently developed single-barrier adsorption model, which is used here as a mean of quantifying the differences in the adsorption between different sequences. In addition, we investigate the effect of DNA hybridization and the presence of mismatch pairing defects and find that flawless hybridization to a complementary oligomer significantly decreases the unbinding force but mismatched hybridization has no obvious effect on it. These results can help optimize a recently envisaged SMFS-based biosensing technology for label-free DNA detection.

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

Breaking point: The unbinding behavior of single supramolecular polymers formed through quadruple hydrogen bonds between 2-ureido-4(1H)-pyrimidinone (UPy) units was studied by AFM-based single-molecule force spectroscopy. The interactions between individual UPy moieties and the stretching of polyethylene glycol (PEG) spacers in reversible supramolecular polymer chains were unveiled by AFM force-displacement measurements.