Robert Walder

Single Molecule Observations of Multiple Protein Populations at the Oil―Water Interface

Single-molecule total internal reflectance fluorescence microscopy was used to observe the dynamic behavior of >4000 bovine serum albumin objects at the silicone oil-water interface. The surface residence time distribution indicated the presence of three populations at the interface. Each population had a characteristic fluorescence intensity and distinctive interfacial diffusion behavior. Larger fluorescence intensity correlated with longer residence times and slower diffusion. These combined observations of fluorescence intensity, surface residence time, and interfacial diffusion suggested that the three populations represent monomers, dimers, and trimers respectively.

Dynamics of protein aggregation at the oil–water interface characterized by single molecule TIRF microscopy

Single molecule total internal reflectance fluorescence microscopy was used to observe the dynamic mechanisms of bovine serum albumin layer formation at the silicone oil–water interface. After an initial induction period, the mean diffusion coefficient of protein objects was observed to decrease as a function of exposure time. Simultaneously, the total adsorption rate was observed to decrease; both observations are consistent with protein layer formation. Importantly, the distribution of diffusion coefficients broadened systematically with exposure time, suggesting the presence of surface objects with a wide range of hydrodynamic radii ranging from a few nm (consistent with monomers) to ∼180 nm. The spatial distribution of the adsorption rate, which was initially uniform, gradually became inhomogeneous, also consistent with the existence of large aggregates. The kinetics of protein layer formation was dependent on the bulk protein concentration in the aqueous phase, and a kinetic population balance model was used to explain the nonlinear concentration dependence.

Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50–1000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using an ultrashort (L = 9 μm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-μm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

Directed Nanoparticle Motion on an Interfacial Free Energy Gradient

Using total internal reflection fluorescence microscopy (TIRFM), we have observed the directed motion of 20 nm probe particles on specific regions of surfaces that exhibited strong gradients of hydrophobicity. Patterned surfaces were prepared by selective photodegradation (using a contact photomask) of a hydrophobically modified fused silica surface. The lateral distribution of hydrophobicity was characterized in situ using the selective affinity of amphiphilic probes (i.e., hydrophobic interaction microscopy). Probe particles were observed to move unidirectionally from regions of lower to higher to hydrophobicity over distances of approximately 1 microm when the hydrophobicity gradient was greater than d(cos theta)/dx = 0.05 +/- 0.02 microm(-1), where theta is the water contact angle on the bare surface. Only adsorption events were observed on energetically homogeneous surface regions.

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

Interfacial protein-protein associations.

While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on polyethylene glycol modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface - with areas of high protein density (i.e., strongly interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e., partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e., clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage.

Ultrastable measurement platform: sub-nm drift over hours in 3D at room temperature

Advanced optical traps can probe single molecules with Ångstrom-scale precision, but drift limits the utility of these instruments. To achieve Å-scale stability, a differential measurement scheme between a pair of laser foci was introduced that substantially exceeds the inherent mechanical stability of various types of microscopes at room temperature. By using lock-in detection to measure both lasers with a single quadrant photodiode, we enhanced the differential stability of this optical reference frame and thereby stabilized an optical-trapping microscope to 0.2 Å laterally over 100 s based on the Allan deviation. In three dimensions, we achieved stabilities of 1 Å over 1,000 s and 1 nm over 15 h. This stability was complemented by high measurement bandwidth (100 kHz). Overall, our compact back-scattered detection enables an ultrastable measurement platform compatible with optical traps, atomic force microscopy, and optical microscopy, including super-resolution techniques.

Stokes–Einstein and desorption-mediated diffusion of protein molecules at the oil–water interface

We observe the diffusive trajectories of individual bovine serum albumin molecules at the oil–water interface over more than two orders of magnitude of oil viscosity. At low oil viscosities, the molecular mobility is dominated by the expected Stokes–Einstein mode of interfacial diffusion, where the effective diffusion coefficient is inversely proportional to both the protein hydrodynamic radius and the oil viscosity. However, for high viscosity oils, a different diffusive regime is observed, with anomalously large effective diffusion coefficients that are relatively insensitive to the oil viscosity and are similar to the measured diffusion coefficient of the same protein at a hydrophobic solid–liquid interface. We propose that in this regime of high oil viscosity, interfacial diffusion is dominated by desorption-mediated diffusion, which also dominates at many solid–liquid interfaces.

Going Vertical To Improve the Accuracy of Atomic Force Microscopy Based Single-Molecule Force Spectroscopy

Single-molecule force spectroscopy (SMFS) is a powerful technique to characterize the energy landscape of individual proteins, the mechanical properties of nucleic acids, and the strength of receptor-ligand interactions. Atomic force microscopy (AFM)-based SMFS benefits from ongoing progress in improving the precision and stability of cantilevers and the AFM itself. Underappreciated is that the accuracy of such AFM studies remains hindered by inadvertently stretching molecules at an angle while measuring only the vertical component of the force and extension, degrading both measurements. This inaccuracy is particularly problematic in AFM studies using double-stranded DNA and RNA due to their large persistence length (p ≈ 50 nm), often limiting such studies to other SMFS platforms (e.g., custom-built optical and magnetic tweezers). Here, we developed an automated algorithm that aligns the AFM tip above the DNAs attachment point to a coverslip. Importantly, this algorithm was performed at low force (10-20 pN) and relatively fast (15-25 s), preserving the connection between the tip and the target molecule. Our data revealed large uncorrected lateral offsets for 100 and 650 nm DNA molecules [24 ± 18 nm (mean ± standard deviation) and 180 ± 110 nm, respectively]. Correcting this offset yielded a 3-fold improvement in accuracy and precision when characterizing DNAs overstretching transition. We also demonstrated high throughput by acquiring 88 geometrically corrected force-extension curves of a single individual 100 nm DNA molecule in ∼40 min and versatility by aligning polyprotein- and PEG-based protein-ligand assays. Importantly, our software-based algorithm was implemented on a commercial AFM, so it can be broadly adopted. More generally, this work illustrates how to enhance AFM-based SMFS by developing more sophisticated data-acquisition protocols.

Improved Free-Energy Landscape Quantification Illustrated with a Computationally Designed Protein-Ligand Interaction

Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (koff ) and the distance to the transition state (Δx≠ ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin. Enhanced data quality enabled an analysis that yielded the height of the transition state (ΔG≠ =6.3±0.2 kcal mol-1 ) and the shape of the energy barrier at the transition state (linear-cubic) in addition to the traditional parameters [koff (=4±0.1×10-4  s-1 ) and Δx≠ (=8.3±0.1 Å)]. We expect this automated and relatively rapid assay to provide a more complete energy landscape description of protein-ligand interactions and, more broadly, the diverse systems studied by AFM-based force spectroscopy.