Senli Guo

Effects of Multiple-Bond Ruptures on Kinetic Parameters Extracted from Force Spectroscopy Measurements: Revisiting Biotin-Streptavidin Interactions

Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe. It is shown that the measured distributions of rupture forces still contain high forces that cannot be described by the forced dissociation from a deep potential well. We employed a recently developed analytical model of simultaneous rupture of two bonds connected by polymer tethers with uneven length to fit the measured distributions. The resulting kinetic parameters agree with the energy landscape predicted by molecular dynamics simulations. It is demonstrated that when more than one molecular bond might rupture during the pulling measurements there is a noise-limited range of probe velocities where the kinetic parameters measured by force spectroscopy correspond to the true energy landscape. Outside this range of velocities, the kinetic parameters extracted by using the standard most probable force approach might be interpreted as artificial energy barriers that are not present in the actual energy landscape. Factors that affect the range of useful velocities are discussed.

Investigation of mechanical properties of insulin crystals by atomic force microscopy.

Mechanical properties of protein crystals and aggregates depend on the conformational and structural properties of individual protein molecules as well as on the packing density and structure within solid materials. An atomic force microscopy (AFM)-based approach is developed to measure the elastic modulus of small protein crystals by nanoindentation and is applied to measure the elasticity of insulin crystals. The top face of the crystals deposited on mica substrates is identified as the (001) face. Insulin crystals exhibit a nearly elastic response during the compression cycle. The elastic modulus measured on the top face has asymmetric distribution with a significant width. This width is related to the uncertainty in the deflection sensitivity. A model that takes into account the distribution of the sensitivity values is used to correct the elastic modulus. Measurements performed in aqueous buffer on several crystals at different locations with three different AFM probes give a mean elastic modulus of 164 +/- 10 MPa. This value is close to the static elastic moduli of other protein crystals measured by different techniques that are usually measured in the range from 100 MPa to 1 GPa. The measured modulus of insulin crystals falls between the elastic modulus values of insulin amyloid fibrils measured previously at two orthogonal directions (a modulus of 14 MPa was measured by compressing the fibril in the direction perpendicular to the fibril axis, and a modulus of 3.3 GPa was measured in the direction along the fibril axis). This comparison indicates the heterogeneous structure of fibrils in the direction perpendicular to the fibril axis, with a packing density of the amyloid fibril core that is higher than the average packing density in insulin crystals. The mechanical wear of insulin crystals is detected during AFM measurements. In nanoindentation experiments on insulin crystal, the compressive load by the AFM tip ( approximately 1 nN, corresponding to a pressure of around 5 MPa) occasionally removes protein molecules from the top or the second top layer of insulin crystal in a sequential manner. The molecular model of this surface damage is proposed. In addition, the removal of the multiple layers of molecules is observed during the AC-mode imaging in aqueous buffer. The number of removed layers depends on the scan size.

Surface elastic properties of human retinal pigment epithelium melanosomes.

Atomic force microscope (AFM) imaging and nanoindentation measurements in water were used to probe the mechanical properties of retinal pigment epithelium melanosomes isolated from 14‐year‐old and 76‐year‐old donors. Topographic imaging reveals surface roughness similar to previous measurements on dry melanosomes. Force‐indentation measurements show different types of responses that were catalogued into four different categories. In these measurements no permanent surface damage of melanosomes was observed as revealed by imaging before and after indentation measurements. The indentation measurements that exhibited nearly elastic responses were used to determine the Young’s modulus of melanosomes. The average Young’s modulus values are similar for 14‐year‐old and 76‐year‐old melanosomes with a somewhat narrower distribution for the 14‐year‐old sample. These elastic modulus values are considerably higher than the modulus of organelles with cytoplasm (<1 MPa) and approaching values of the modulus of protein crystals (∼100 MPa) indicating rather high packing density of biologic material in melanosomes. The width of the Young’s modulus distributions is considerable spanning from few megapascals to few tens of megapascals indicating large heterogeneity in the structure. A fraction of the force curves cannot be described by the homogeneous elastic sample model; these force curves are consistent with ∼10 nm structural heterogeneity in melanosomes. The approach‐withdraw hysteresis indicates a significant viscoelasticity, particularly in the samples from the 14‐year‐old sample. Adhesion of the AFM probe was detected on ∼3% and ∼20% of the surface of 14‐year‐old and 76‐year‐old samples, respectively. In light of previous studies on these same melanosomes using photoelectron emission microscopy, this adhesion is attributed to the presence of lipofuscin on the surface of the melanosomes. This suggestion indicates that part of the difference in photochemical properties between the old and young melanosomes originates from surface lipofuscin.

Association kinetics from single molecule force spectroscopy measurements.

Single molecule force spectroscopy is often used to study the dissociation of single molecules by applying mechanical force to the intermolecular bond. These measurements provide the kinetic parameters of dissociation. We present what to our knowledge is a new atomic force microscopy-based approach to obtain the activation energy of the association reaction and approximate grafting density of reactive receptors using the dependence of the probability to form molecular bonds on probe velocity when one of the interacting molecules is tethered by a flexible polymeric linker to the atomic force microscopy probe. Possible errors in the activation energy measured with this approach are considered and resulting corrections are included in the data analysis. This new approach uses the same experimental setup as traditional force spectroscopy measurements that quantify dissociation kinetics. We apply the developed methodology to measure the activation energy of biotin-streptavidin association (including a contribution from the steric factor) and obtain a value of 8 +/- 1 kT. This value is consistent with the association rate measured previously in solution. Comparison with the solution-derived activation energy indicates that kinetics of biotin-streptavidin binding is mainly controlled by the reaction step.

Mechanical Distortion of Protein Receptor Decreases the Lifetime of a Receptor-Ligand Bond

Substantial experimental evidence indicates that the mechanical force applied to pull apart non-covalent molecular bonds (such as receptor-ligand pairs) can significantly decrease the bond lifetime. This evidence is often generated in single-molecule experiments that are designed to specifically test effects of pulling forces. However, the effect of compressive forces on the lifetime of receptor-ligand bonds remains largely unexplored. Here we extend the common usage of the atomic force microscopy technique to study whether compressive forces applied to bound streptavidin-biotin species can significantly accelerate the rate of dissociation. Presented experimental data indicate that compressive forces can substantially decrease the lifetime of the molecular bond. Surprisingly, the efficiency of accelerating dissociation by compressive forces sometimes exceeds the enhancement of the dissociation rate measured in pulling experiments, indicating that compressive forces applied to the bound species might be efficiently used to control the lifetime of adhesion bonds.

Kinetic Parameters from Detection Probability in Single Molecule Force Spectroscopy

The detection probability of rupture events in AFM force spectroscopy measurements presents a viable alternative to standard methods for extracting kinetic parameters of dissociation. The detection probability has a maximum as a function of the probe velocity where (1) the probability to form a molecular bond is independent of the probe velocity and (2) the detection of rupture events is limited by noise and performed with a constant density of data points per distance of the probe displacement. This newly developed model indicates that the optimal detection velocity is independent of dissociation rate and depends on the distance to the barrier kinetic parameter. Therefore, the kinetic parameters of bond dissociation can be extracted from the dependence of detection probability on probe velocity and the detection threshold. This approach is sensitive to low rupture forces and therefore is complementary to the common most probable force data analysis approach. The developed approach is tested using rupture forces measured with specific bonds between biotin and streptavidin and with nonspecific bonds between linear alkanes in water. Results for the analysis of specific bonds rupture are consistent with the previous measurements, suggesting that rupture forces spanning a wide range of values originate from the same binding potential. Kinetic parameters obtained for linear alkanes are significantly different from previous measurements suggesting possible heterogeneity of the bound state.

Apparent Dependence of Rupture Force on Loading Rate in Single-Molecule Force Spectroscopy

Force spectroscopy is becoming an important tool in physicochemical research and allows the extraction of kinetic parameters of dissociation that are not available to other techniques. The theory of single-molecule force spectroscopy has received considerable attention over the last several years and continues to be developed. Atomic force microscopy (AFM) in the force measurement mode is one of the most common methods to perform force spectroscopy measurements. In these experiments, rupture forces between interacting molecules are measured as a function of the AFM probe velocity. The resulting dependence of most probable rupture force on loading rate is then used to extract kinetic information. Although this technique is becoming widespread, we find that some important experimental aspects require more thorough consideration. One of these seemingly simple aspects is a methodology of extracting rupture force from a force curve. Herein, we discuss how this methodology might have a significant impact on the interpretation of force spectroscopy measurements. Consider the force-separation curve with a rupture event shown in Figure 1. A part of the force curve corresponding to the stretching of the polymeric tether prior to the rupture without added noise is shown with a solid line. With noise this dependence might show a considerable amount of scatter. Therefore, if the rupture force is obtained as a difference between the most outstanding data points in the vicinity of the rupture event, then the root-mean-square (RMS) amplitude of the noise will be added to the rupture force, as illustrated in the figure. The RMS amplitude of noise that is added might depend on the pulling velocity and therefore affect kinetic parameters obtained in force spectroscopy measurements. The dependence of the noise amplitude Fn the on probe velocity v can be written as [Eq. (1)]: