Single molecule force spectroscopy using polyproteins

In recent years single molecule force spectroscopy has emerged as a powerful new tool to explore the mechanical stability and folding pathways of individual proteins. This technique is used to apply a stretching force between two points of a protein, unfolding the protein to an extended state. By measuring the unfolding and folding trajectories of individual proteins, insight can be gained into the physical mechanisms of protein folding. In this tutorial review we introduce the reader to single molecule force spectroscopy using the atomic force microscope (AFM), and explain the two main modes of operation of the AFM for force spectroscopy: force-extension and force-clamp. We introduce the approach of using polyproteins to obtain a clear mechanical fingerprint for monitoring the response of proteins to an applied mechanical force. In addition, we provide an informative and representative review of recent research on proteins using single molecule force spectroscopy. We focus on areas which have made a significant contribution to the single molecule protein folding field and highlight emerging areas of research which have wider implications for the general scientific community.     

Single molecule force spectroscopy of asphaltene aggregates

Asphaltene aggregation and deposition cause severe problems in nearly all phases of petroleum processing. To resolve those problems, understanding the aggregation mechanisms is a prerequisite and has attracted the interest of a great number of investigators. However, to date, the nature and extent of asphaltene aggregation remain widely debated. In the present study, we attempt to investigate asphaltene aggregation from a completely new perspective. The technique of single molecule force spectroscopy (SMFS) was used to investigate the response of single asphaltene aggregates under an external pulling force. Force curves representing the stretching of single asphaltene aggregates were obtained in simple electrolyte solutions (KCl and calcium) and organic solvents (toluene and heptane). These force curves were well-fitted by the modified worm-like chain model, indicating that those asphaltene aggregates acted like long-chain polymers under pulling by an external force. It was found that lower solution pH values and the presence of divalent cations resulted in a lower bending rigidity of the formed aggregates. The information retrieved from the force curves suggests that asphaltene molecules with a structure featuring small aromatic clusters connected by aliphatic chains do exist and that asphaltene aggregation could occur through a linear polymerization mechanism. The current study extends the application scope of SMFS.     

Single molecule atomic force microscopy and force spectroscopy of chitosan

Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10(-20)J, 1.8±0.3×10(-20)J and 3.5±0.3×10(-20)J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications.     

Single molecule force spectroscopy on poly(vinyl alcohol) by atomic force microscopy

The elastic properties of poly(vinyl alcohol) (PVA) were investigated on the nanoscale using the new technique of single molecule force spectroscopy by atomic force microscopy (AFM). It was found that the elastic properties of PVA molecules scale linearly with their contour lengths. This finding corroborates that the deformation of individual PVA chains is measured. The force spectra of PVA show a kink at around 200 pN and cannot be fitted by an extended Langevin function. The deviation of the elastic behavior of PVA from a freely jointed chain model may indicate the presence of a suprastructure of PVA in NaCl solution.     

Investigation on the folding mode of a polymer chain in a spiral crystal by single molecule force spectroscopy

Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy （AFM） imaging and force spectroscopy to pull a single polyethylene oxide （PEO） chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.     

Single molecule modulation spectroscopy of conjugated polymers

This paper describes a new single molecule spectroscopy approach for the investigation of triplet-triplet and singlet-triplet interactions in conjugated polymers. The technique involves the irradiation of isolated single, mulitchromophoric, conjugated polymer molecules by a repetitive sequence of variable-intensity microsecond time scale excitation pulses. The fluorescence intensity is synchronously time-averaged for thousands of cycles of the pulse sequence to yield a high signal-to-noise fluorescence transient on the microsecond time scale. The transient can be analyzed with kinetic models to obtain quantitative information about the kinetics of triplet-triplet exciton annihilation and the quenching of singlet excitons by triplet excitons in conjugated polymers.     

Direct detection of the formation of V-amylose helix by single molecule force spectroscopy

An important polysaccharide, amylose crystallizes as a regular single left-handed helix from a propanol, butanol, or iodine solution. However, its solution structure remains elusive because amylose does not form molecular solutions in these solvents, and standard spectroscopic techniques cannot be exploited to determine its structure. Using AFM, we forced individual amylose chains adsorbed to a surface to enter these poor solvents and carried out stretch-release measurements on them in solution. In this manner, we directly captured the formation of individual amylose helices induced by butanol and iodine. With an accuracy approaching that of X-ray diffraction on amylose crystals, we determined that the pitch of the helix in solution is 1.3 angstroms/ring. We also directly measured the force driving the formation of the helix in solution to be 50 pN. SMD simulations in explicit butanol reproduced the AFM-measured force-extension curves and revealed that the long plateau feature is caused by the rupture of O(2)n-O(6)(n+6) and O(3)n-O(6)(n+6) hydrogen bonds and by the unwinding of the helix. We also found that amylose helices formed in iodine solution are more compliant and hysteretic as compared to helices in butanol, which extend/relax reversibly. In iodine solution, the formation of the helix is inhibited by force and limited by the slow kinetics of the amylose-iodine complex. By forcing individual molecules into poor solvents and performing force spectroscopy measurements in solution, our AFM approach uniquely supplements X-ray diffraction and NMR methods for investigating solution conformations of insoluble biopolymers.     

Single molecule spectroscopy. Perylene in the Shpol''skii matrix n-nonane

Single perylene molecules in the Shpol'skii matrix n-nonane have been investigated at 1.7 K using a frequency-doubled cw Ti: sapphire laser as an excitation source. Fluorescence excitation spectra within the inhomogeneously broadened 0-0 absorption band around 443.8 nm were taken. The combination of high fluorescence quatumm yield and low intersystem crossing rate with a relatively short-lived triplet state allowed the direct observation of fluorescence photon bunches. The time distribution of the resulting emission gaps for the specific single molecule studied in detail leads to a triplet lifetime of τ_p = 1.1 ± 0.5 ms.     

Single molecule spectroscopy studies of diffusion in mesoporous silica thin films

Single molecule spectroscopy is applied in studies of diffusion and surface adsorption in sol-gel-derived mesoporous silica thin films. Mesoporous films are obtained by spin casting surfactant-templated sols onto glass substrates. Small-angle X-ray diffraction results are consistent with hexagonally ordered mesophases in as-synthesized (i.e., surfactant-containing) films. Upon calcination, a 30% contraction and disordering of these structures occurs. Nile Red is used as a fluorescent probe of both the as-synthesized and calcined films. It is loaded into the samples at subnanomolar levels either prior to spin casting or after calcination. Fluorescence imaging and single-point fluorescence time transients show the dye molecules to be relatively mobile in the as-synthesized samples. In contrast, the molecules appear entrapped at fixed locations in dry calcined films. In calcined films rehydrated under high humidity conditions, the Nile Red molecules again become mobile. Time transients obtained from the as-synthesized and rehydrated samples provide clear evidence for frequent reversible adsorption of the dye to the silica surfaces. Autocorrelations of the time transients provide quantitative data on the mean diffusion coefficients (D = 2.4 x 10(-10) and 2.6 x 10(-10) cm2/s) and mean desorption times (1/k = 25 and 40 s) for the as-synthesized and rehydrated films, respectively. The results prove both water and surfactant play important roles in governing matrix interactions and mass transport.     

Single molecule spectroscopy of conjugated polymer chains in an electric field-aligned liquid crystal

Using single molecule polarization spectroscopy, we investigated the alignment of a polymer solute with respect to the liquid crystal (LC) director in an LC device while applying an external electric field. The polymer solute is poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (or MEH-PPV), and the LC solvent is 5CB. The electric field induces a change in the LC director orientation from a planar alignment (no electric field) to a perpendicular (homeotropic) alignment with an applied field of 5.5 x 103 V/cm. We find that the polymer chains align with the LC director in both planar and homeotropic alignment when measured in the bulk of the LC solution away from the device interface. Single molecule polarization distributions measured as a function of distance from the LC device interface reveal a continuous change of the MEH-PPV alignment from planar to homeotropic. The observed polarization distributions are modeled using a conventional elastic model that predicts the depth profile of the LC director orientation for the applied electric field. The excellent agreement between experiment and simulations shows that the alignment of MEH-PPV follows the LC director throughout the LC sample. Furthermore, our results suggest that conjugated polymers such as MEH-PPV can be used as sensitive local probes to explore complex (and unknown) structures in anisotropic media.     

Perfluorophenyl azide immobilization chemistry for single molecule force spectroscopy of the concanavalin A/mannose interaction

The versatility of perfluorophenyl azide (PFPA) derivatives makes them useful for attaching a wide variety of biomolecules and polymers to surfaces. Herein, a single molecule force spectroscopy (SMFS) study of the concanavalin A/mannose interaction was carried out using PFPA immobilization chemistry. SMFS of the concanavalin A/mannose interaction yielded an average unbinding force of 70-80 pN for loading rates between 8000 and 40,000 pN/s for mannose surfaces on aminated glass, and an unbinding force of 57 ± 20 pN at 6960 pN/s for mannose surfaces on gold-coated glass. Dynamic force spectroscopy was used to determine the dissociation rate constant, k(off), for this interaction to be 0.16 s(-1).     

Sub-doppler spectroscopy of the NaCs molecule

Doppler-free polarization spectroscope in combination with double-resonance techniques and laser-induced fluorescence spectroscope has been used to det     

Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe

The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R(2)). Cells that contain an R(2) ≥ 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies.     

Single molecule tracking analysis reveals that the surface mobility of amyloid oligomers is driven by their conformational structure

Several models have been proposed to explain the cytotoxicity of Aβ oligomers. The structural polymorphism of the oligomers can account for the various toxic effects observed. By combining the use of conformation-specific antibodies and single particle tracking techniques, we have investigated the mobility of individual Aβ1-42 oligomers on the plasma membrane of living cells. Distinct structural types of Aβ1-42 oligomers were labeled with two different conformation-specific antibodies. While both types of oligomers showed a heterogeneous dynamic behavior, their overall mobility was found to be significantly different. Conversely, we discovered that other amyloid oligomers sharing a similar conformation but composed of different peptides (amylin and prion Sup35NM) display dynamic behaviors comparable to those found for Aβ1-42 oligomers. This study provides evidence for a link between the quaternary structure and the membrane mobility of proteins, revealing that structurally analogous supramolecular assemblies diffuse similarly in cells.     

Single site electronic spectroscopy of palladium chlorin in n-octane matrixes at 7K

The high resolution, single site emission and absorption spectra of palladium chlorin (PdC) in n-octane matrixes at 7K are reported. The emission and Q and Soret band absorption regions were investigated. The vibrational frequencies of the ground and the lowest energy pipi* excited states were determined from luminescence and excitation spectra, respectively. The emission from PdC was entirely phosphorescence. The emission and Qy region spectra of the complex are similar, having intense, narrow origin bands followed by relatively weak but orderly vibrational structure. The Qx region of this metal chlorin does not have a clear origin and exhibits complex vibrational structure which increases in intensity going to higher energy. In the Soret region of PdC there is only a single intense, broad band.