Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations.

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to µm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α3D using atomic force microscopy (AFM)-based force spectroscopy. α3D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α3D's configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy's ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α3D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam-modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 kBT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Dressed Rabi Oscillation in a Crystalline Organic Radical.

Free electron laser-powered pulsed electron paramagnetic resonance experiments performed at 240 GHz/8.56 T on the crystalline organic radical 1,3-bisdiphenylene-2-phenylallyl reveal a tip-angle dependent resonant frequency. Frequency shifts as large as 11 MHz (45 ppm) are observed during a single Rabi oscillation. We attribute the frequency shifts to a "dressing" of the nutation by spin-spin interactions. A nonlinear semiclassical model which includes a temperature- and sample-geometry-dependent demagnetizing field reproduces experimental results. Because experiments are performed without a cavity, radiation damping, the most common nonlinear interaction in magnetic resonance, is negligible in our experiments.

Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization.

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high-precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near-equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 µs resolution. The third of these states was retinal-stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native-like conditions.

Force Spectroscopy with 9-µs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability. Recently, we modified commercial cantilevers with a focused ion beam to optimize their properties for SMFS. Here, we extend this capability by modifying a 40 × 18 µm2 cantilever into one terminated with a gold-coated, 4 × 4 µm2 reflective region connected to an uncoated 2-µm-wide central shaft. This "Warhammer" geometry achieved 8.5-µs resolution coupled with improved force precision and sub-pN stability over 100 s when measured on a commercial AFM. We highlighted this cantilever's biological utility by first resolving a calmodulin unfolding intermediate previously undetected by AFM and then measuring the stabilization of calmodulin by myosin light chain kinase at dramatically higher unfolding velocities than in previous AFM studies. More generally, enhancing data quality via an improved combination of time resolution, force precision, and force stability will broadly benefit biological applications of AFM.

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.

Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins.

Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates-many separated by as few as two or three amino acids-exhibited complex dynamics, including frequent refolding and state occupancies of <10 µs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.

Optimizing force spectroscopy by modifying commercial cantilevers: Improved stability, precision, and temporal resolution.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing data quality via improved force stability, force precision, and temporal resolution. For an advanced, small-format commercial AFM, we illustrate how these three metrics are limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf=0.01-20Hz) is routinely achieved by removing a long (L=100µm) cantilever's gold coating. Next, this sub-pN bandwidth is extended by a factor of ∼50 to span five decades of bandwidth (Δf=0.01-1000Hz) by using a focused ion beam (FIB) to modify a shorter (L=40µm) cantilever. Finally, FIB-modifying an ultrashort (L=9µm) cantilever improves its force stability and precision while maintaining 1-µs temporal resolution. These modified ultrashort cantilevers have a reduced quality factor (Q≈0.5) and therefore do not apply a substantial (30-90pN), high-frequency force modulation to the molecule, a phenomenon that is unaccounted for in traditional SMFS analysis. Currently, there is no perfect cantilever for all applications. Optimizing AFM-based SMFS requires understanding the tradeoffs inherent to using a specific cantilever and choosing the one best suited to a particular application.

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.

Determining the oligomeric structure of proteorhodopsin by Gd3+ -based pulsed dipolar spectroscopy of multiple distances.

The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

Effect of electron spin dynamics on solid-state dynamic nuclear polarization performance.

For the broadest dissemination of solid-state dynamic nuclear polarization (ssDNP) enhanced NMR as a material characterization tool, the ability to employ generic mono-nitroxide radicals as spin probes is critical. A better understanding of the factors contributing to ssDNP efficiency is needed to rationally optimize the experimental condition for the practically accessible spin probes at hand. This study seeks to advance the mechanistic understanding of ssDNP by examining the effect of electron spin dynamics on ssDNP performance at liquid helium temperatures (4-40 K). The key observation is that bi-radicals and mono-radicals can generate comparable nuclear spin polarization at 4 K and 7 T, which is in contrast to the observation for ssDNP at liquid nitrogen temperatures (80-150 K) that finds bi-radicals to clearly outperform mono-radicals. To rationalize this observation, we analyze the change in the DNP-induced nuclear spin polarization (Pn) and the characteristic ssDNP signal buildup time as a function of electron spin relaxation rates that are modulated by the mono- and bi-radical spin concentration. Changes in Pn are consistent with a systematic variation in the product of the electron spin-lattice relaxation time and the electron spin flip-flop rate that constitutes an integral saturation factor of an inhomogeneously broadened EPR spectrum. We show that the comparable Pn achieved with both radical species can be reconciled with a comparable integral EPR saturation factor. Surprisingly, the largest Pn is observed at an intermediate spin concentration for both mono- and bi-radicals. At the highest radical concentration, the stronger inter-electron spin dipolar coupling favors ssDNP, while oversaturation diminishes Pn, as experimentally verified by the observation of a maximum Pn at an intermediate, not the maximum, microwave (µw) power. At the maximum µw power, oversaturation reduces the electron spin population differential that must be upheld between electron spins that span a frequency difference matching the (1)H NMR frequency-characteristic of the cross effect DNP. This new mechanistic insight allows us to rationalize experimental conditions where generic mono-nitroxide probes can offer competitive ssDNP performance to that of custom designed bi-radicals, and thus helps to vastly expand the application scope of ssDNP for the study of functional materials and solids.

Temperature dependence of high field 13C dynamic nuclear polarization processes with trityl radicals below 35 Kelvin.

In order to facilitate versatile applications with high field dynamic nuclear polarization (DNP), it is important to be able to optimize the DNP performance, i.e. reach high nuclear hyperpolarization within a short signal build up time. Given that the solid-state DNP process is strongly temperature-dependent, it is important to benchmark the temperature dependence of various DNP and electron paramagnetic resonance (EPR) parameters that can then be used to test and develop theories and models for high field DNP mechanisms. However, DNP and EPR experiments at high fields and cryogenic temperatures below 20 Kelvin usually require home built instrumentation, and therefore even basic experimental observations are lacking in the literature. DNP and EPR experiments at 7 T (197 GHz) and 8.5 T (240 GHz), respectively, were conducted at temperatures between 35 K and 3.7 K where the electron thermal polarization changes from 13.4% to 85.6%, respectively. The samples are frozen solutions of 15 mM OX063Me trityl radicals in various mixtures of [1-(13)C]pyruvic acid, glycerol, and Gd(3+)-chelates. For all sample mixtures, the trityl EPR lines are found to be inhomogeneously broadened and the dominant DNP mechanism is shown to be the cross effect (CE). A 20%, 11%, and 6.77% (13)C polarization is achieved at 3.7 K with a [1-(13)C]pyruvic-glycerol-H2O sample, the addition of 2 mM of Gd(3+)-chelates, and pure [1-(13)C]pyruvic acid, respectively. When T1n is sufficiently long, our results seem to suggest T1e is a key variable in the DNP process, where longer T1e values correlate with larger DNP enhancements (εDNP). The experimental data reported here on the temperature dependence of T1n, T1e, Tm (electron phase memory time), the EPR linewidth, TDNP and ε(DNP) at high fields will be helpful for testing the mechanism and theory of DNP processes.

Extending the distance range accessed with continuous wave EPR with Gd3+ spin probes at high magnetic fields.

Interspin distances between 0.8 nm and 2.0 nm can be measured through the dipolar broadening of the continuous wave (cw) EPR spectrum of nitroxide spin labels at X-band (9.4 GHz, 0.35 T). We introduce Gd(3+) as a promising alternative spin label for distance measurements by cw EPR above 7 Tesla, where the |-1/2〉 to |1/2〉 transition narrows below 1 mT and becomes extremely sensitive to dipolar broadening. To estimate the distance limits of cw EPR with Gd(3+), we have measured spectra of frozen solutions of GdCl3 at 8.6 T (240 GHz) and 10 K at concentrations ranging from 50 mM to 0.1 mM, covering a range of average interspin distances. These experiments show substantial dipolar broadening at distances where line broadening cannot be observed with nitroxides at X-band. This data, and its agreement with calculated dipolar-broadened lineshapes, show Gd(3+) to be sensitive to distances as long as ∼3.8 nm. Further, the linewidth of a bis-Gd(3+) complex with a flexible ∼1.6 nm bridge is strongly broadened as compared to the mono-Gd(3+) complex, demonstrating the potential for application to pairwise distances. Gd-DOTA-based chelates that can be functionalized to protein surfaces display linewidths narrower than aqueous GdCl3, implying they should be even more sensitive to dipolar broadening. Therefore, we suggest that the combination of tailored Gd(3+) labels and high magnetic fields can extend the longest interspin distances measurable by cw EPR from 2.0 nm to 3.8 nm. cw EPR data at 260 K demonstrate that the line broadening remains clear out to similar average interspin distances, offering Gd(3+) probes as promising distance rulers at temperatures higher than possible with conventional pulsed EPR distance measurements.

Phase cycling with a 240 GHz, free electron laser-powered electron paramagnetic resonance spectrometer.

Electron paramagnetic resonance (EPR) powered by a free electron laser (FEL) has been shown to dramatically expand the capabilities of EPR at frequencies above ~100 GHz, where other high-power sources are unavailable. High-power pulses are necessary to achieve fast (<10 ns) spin rotations in order to alleviate the limited excitation bandwidth and time resolution that typically hamper pulsed EPR at these high frequencies. While at these frequencies, an FEL is the only source that provides ~1 kW of power and can be tuned continuously up to frequencies above 1 THz, it has only recently been implemented for one- and two-pulse EPR, and the capabilities of the FEL as an EPR source are still being expanded. This manuscript presents phase cycling of two pulses in an FEL-EPR spectrometer operating at 240 GHz. Given that the FEL, unlike amplifiers, cannot be easily phase-locked to a reference source, we instead apply retrospective data processing to measure the relative phase of each FEL pulse in order to correct the signal phase accordingly. This allows the measured signal to be averaged coherently, and the randomly changing phase of the FEL pulse results in a stochastic phase cycle, which, in the limit of many pulses, efficiently cancels artifacts and improves sensitivity. Further, the relative phase between the first and second pulse, which originates from the difference in path length traversed by each pulse, can be experimentally measured without phase-sensitive detection. We show that the relative phase of the two pulses can be precisely tuned, as well as distinctly switched by a fixed amount, with the insertion of a dielectric material into the quasi-optical path of one of the pulses. Taken together, these techniques offer many of the advantages of arbitrary phase control, and allow application of phase cycling to dramatically enhance signal quality in pulsed EPR experiments utilizing high-power sources that cannot be phase-locked.

Distance measurements across randomly distributed nitroxide probes from the temperature dependence of the electron spin phase memory time at 240 GHz.

At 8.5 T, the polarization of an ensemble of electron spins is essentially 100% at 2 K, and decreases to 30% at 20 K. The strong temperature dependence of the electron spin polarization between 2 and 20 K leads to the phenomenon of spin bath quenching: temporal fluctuations of the dipolar magnetic fields associated with the energy-conserving spin "flip-flop" process are quenched as the temperature of the spin bath is lowered to the point of nearly complete spin polarization. This work uses pulsed electron paramagnetic resonance (EPR) at 240 GHz to investigate the effects of spin bath quenching on the phase memory times (T(M)) of randomly-distributed ensembles of nitroxide molecules below 20 K at 8.5 T. For a given electron spin concentration, a characteristic, dipolar flip-flop rate (W) is extracted by fitting the temperature dependence of T(M) to a simple model of decoherence driven by the spin flip-flop process. In frozen solutions of 4-Amino-TEMPO, a stable nitroxide radical in a deuterated water-glass, a calibration is used to quantify average spin-spin distances as large as r=6.6 nm from the dipolar flip-flop rate. For longer distances, nuclear spin fluctuations, which are not frozen out, begin to dominate over the electron spin flip-flop processes, placing an effective ceiling on this method for nitroxide molecules. For a bulk solution with a three-dimensional distribution of nitroxide molecules at concentration n, we find W∝n∝1/r(3), which is consistent with magnetic dipolar spin interactions. Alternatively, we observe W∝n(32) for nitroxides tethered to a quasi two-dimensional surface of large (Ø∼200 nm), unilamellar, lipid vesicles, demonstrating that the quantification of spin bath quenching can also be used to discern the geometry of molecular assembly or organization.

A 200 GHz dynamic nuclear polarization spectrometer.

We present our experimental setup for both dynamic nuclear polarization (DNP) and electron paramagnetic resonance (EPR) detection at 7 T using a quasi-optical bridge for propagation of the 200 GHz beam and our initial results obtained at 4 K. Our quasi-optical bridge allows the polarization of the microwave beam to be changed from linear to circular. Only the handedness of circular polarization in the direction of the Larmor precession is absorbed by the electron spins, so a gain in effective microwave power of two is expected for circular vs. linear polarization. Our results show an increase in DNP signal enhancement of 28% when using circularly vs. linearly polarized radiation. We measured a maximum signal enhancement of 65 times that of thermal polarization for a (13)C labeled urea sample corresponding to 3% nuclear spin polarization. Since the time constant for nuclear spin polarization buildup during microwave irradiation is 10 times faster than the (13)C nuclear spin T(1), the actual gain in detection sensitivity with DNP is much greater.