Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy.

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy-based single-molecule force spectroscopy. Precisely, timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR's G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the "open" portion of bR's photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR's open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins.

Dissociation of polyethylene liners with the Depuy Pinnacle cup: a report of 26 cases.

BACKGROUND: Although the Pinnacle Acetabular Hip System (DePuy Synthes) has demonstrated excellent survivorship results since it was first introduced in 2003, there have been a growing number of cases indicating that Pinnacle liners may be subject to a higher-than-expected rate of early dissociation failure. Between 2006 and 2020, our Centre received 212 retrieved Pinnacle liners from Western Australian hospitals. Of these, 26 were removed due to liner dissociation. METHODS: To better understand the frequency and cause of this complication we assessed all retrieved Pinnacle acetabular components for type, damage modes and patient demographics. The leverage force required to dissociate Pinnacle liners was also measured and compared with another commonly used acetabular system, the Trident (Stryker Orthopaedics). RESULTS: The estimated minimum incidence of liner dissociation from our data was 0.35%. Characterisation of dissociated Pinnacle cases (n = 26) revealed 73% were female with an average age of 59 compared to all retrieved Pinnacle cases (n = 212) where 58% were female with an average age of 66. Retrieval analysis indicated plastic deformation of the liner into an ovoid shape, signs of impingement on the rim postero-superiorly and shearing of the liner's anti-rotation tabs was common. Mechanical testing indicated that the dissociation strength of Pinnacle cups decreases at approximately 6.6 N/year in situ (p = 0.01). CONCLUSION: The survival rate of Pinnacle acetabular cups is exceptional with only 5% revised at 10 years. However, surgeons should be aware of the clinical symptoms and high-risk demographics when assessing patients with polyethylene Pinnacle liners.

Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay.

Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently. To do so, we engineered a dsDNA molecule with a designed sequence of specified GC content positioned between two enzymatically induced, site-specific nicks. Partially pulling this substrate into the overstretching transition of DNA (~65 pN) using an optical trap led to controlled dissociation of the ssDNA segment delineated by the two nicks. Here, we describe protocols for generating ssDNA of up to 1000 nucleotides as well as more complex structures, such as a 120-base-pair DNA hairpin positioned next to a 33-nucleotide ssDNA segment. The utility of the hairpin substrate was demonstrated by measuring the motion of E. coli. RecQ, a 3'-to-5' DNA helicase.

Feeding a high-energy finishing diet upon arrival to high-risk feedlot calves: effects on health, performance, ruminal pH, rumination, serum metabolites, and carcass traits.

This study evaluated the impacts of feeding a high-energy finishing diet during both the receiving and finishing period compared with a lower-energy receiving diet with adaptation to the finishing diet on health, performance, serum chemistry, ruminal pH, rumination, and carcass characteristics of high-risk feedlot cattle. Five truck-load blocks of steers (n = 101) and bulls (n = 299) were used in a generalized complete block design and randomly assigned to receive: 1) finishing diet for the entire feeding period (FIN) or 2) receiving diet for the first 56 d, followed by a transition to the finishing diet (REC). All cattle were fed ad libitum and consumed the same diet by day 74. A subset of cattle (n = 48) was randomly selected to quantify ruminal pH, temperature, and rumination time. Ultrasound images were collected on days 0, 74, and 146 to determine fat thickness over the 12th rib and rump, and carcass characteristics were determined after slaughter. Cattle fed FIN had less (P < 0.01) dry matter intake (DMI) from days 0 to 74, but DMI did not differ (P = 0.80) after day 74. From days 0 to final, DMI was 0.26 kg less for FIN compared with REC (P = 0.01). However, calculated metabolizable energy intake was not different from days 0 to 74 (P = 0.19), days 74 to final (P = 0.80), or overall (P = 0.78). Body weight (BW) on day 74 was greater (P < 0.01) and final BW tended to be greater (P = 0.10) for FIN compared with REC. Cattle consuming FIN had greater (P < 0.01) average daily gain and increased (P < 0.01) gain:feed from days 0 to 74. There were no differences (P ≥ 0.31) in health outcomes. On day 74, FIN had greater (P = 0.04) fat thickness over the rump and rib but did not differ (P ≥ 0.52) on day 146. Carcasses of FIN had greater (P = 0.04) hot carcass weight with no difference (P ≥ 0.11) in ribeye area, 12th rib fat thickness, yield grade, or quality grade. There was no difference (P = 0.18) in liver abscess rate. There was a diet × day interaction for blood urea nitrogen (P = 0.02) such that concentration decreased from days 0 to 28 in both treatments, but was less on day 28 for FIN. Ruminal pH was greater on days 2 and 61 and rumination time was less from days 0 to 28 for FIN (diet × day interaction; P < 0.01). Overall, these results suggest that providing a finishing diet fed ad libitum to high-risk calves upon arrival may be a viable alternative to a low-energy receiving diet.

When high-risk cattle arrive at the feedlot, they have low feed consumption and a greater risk for disease because of stress, inflammation, and exposure to pathogens. Because of reduced feed consumption, newly arrived cattle may not be able to meet their energy requirement for growth during the first several weeks after feedlot arrival. Therefore, providing a high-energy finishing diet (FIN) when stressed cattle arrive at the feedlot may allow for greater growth performance and improved health when compared with a traditional, low-energy receiving diet that contains more roughage (REC). Our study evaluated this concept and we observed that cattle fed FIN had greater body weight, average daily gain, and gain:feed (G:F) within the first 56 d when the different diets were fed, with no difference in growth performance after the cattle consuming REC transitioned to FIN on day 74. However, cattle consuming FIN had greater hot carcass weight and G:F over the entire feeding period. There were no differences in health outcomes among treatments. Overall, providing a high-energy finishing diet to high-risk cattle upon arrival to the feedlot improved growth performance with no impact on health.

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy.

Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 µs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR's G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule-derived ΔΔG for mutant L223A (-2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (-2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val217 and a natively bound squalene lipid, highlighting the contribution of membrane protein-lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.

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.

Type III secretion system effector proteins are mechanically labile.

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins-such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)-impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy ([Formula: see text]) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published [Formula: see text] for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (Funfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (Funfold > 80 pN) and brittle (Δx‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Correcting molecular transition rates measured by single-molecule force spectroscopy for limited temporal resolution.

Equilibrium free-energy-landscape parameters governing biomolecular folding can be determined from nonequilibrium force-induced unfolding by measuring the rates k for transitioning back and forth between states as a function of force F. However, bias in the observed forward and reverse rates is introduced by limited effective temporal resolution, which includes the mechanical response time of the force probe and any smoothing used to improve the signal-to-noise ratio. Here we use simulations to characterize this bias, which is most prevalent when the ratio of forward and reverse rates is far from unity. We find deviations in k(F) at high rates, due to unobserved transitions from short- to long-lived states, and at low rates, due to the corresponding unobserved transitions from long- to short-lived states. These missing events introduce erroneous curvature in log(k) vs F that leads to incorrect landscape parameter determination. To correct the measured k(F), we derive a pair of model-independent analytical formulas. The first correction accounts for unobserved transitions from short- to long-lived states, but does surprisingly little to correct the erroneous energy-landscape parameters. Only by subsequently applying the second formula, which corrects the corresponding reverse process, do we recover the expected k(F) and energy-landscape quantities. Going forward, these corrections should be applied to transition-rate data whenever the highest measured rate is not at least an order of magnitude slower than the effective temporal resolution.

Quantifying the Native Energetics Stabilizing Bacteriorhodopsin by Single-Molecule Force Spectroscopy.

We quantified the equilibrium (un)folding free energy ΔG_{0} of an eight-amino-acid region starting from the fully folded state of the model membrane-protein bacteriorhodopsin using single-molecule force spectroscopy. Analysis of equilibrium and nonequilibrium data yielded consistent, high-precision determinations of ΔG_{0} via multiple techniques (force-dependent kinetics, Crooks fluctuation theorem, and inverse Boltzmann analysis). We also deduced the full 1D projection of the free-energy landscape in this region. Importantly, ΔG_{0} was determined in bacteriorhodopsin's native bilayer, an advance over traditional results obtained by chemical denaturation in nonphysiological detergent micelles.

Bending and looping of long DNA by Polycomb repressive complex 2 revealed by AFM imaging in liquid.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA's helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2's methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing.

Alzheimer's Diagnosis: Real-World Physician Behavior Across Countries.

INTRODUCTION: Appropriate management of patients with Alzheimer's disease (AD) helps preserve their independence and time at home. We explored physician behavior in the management of AD, focusing on diagnosis. METHODS: Online questionnaires and patient record forms (PRFs) were created by an independent market research agency and completed by participating physicians. Physicians were recruited from France, Germany, Japan, the UK, and the USA. A sample of 1086 physicians was recruited, including general practitioners, geriatricians, neurologists, and psychiatrists. Physicians completed an online interview and 2-3 PRFs based on randomly selected records of their patients with AD. Data on triggers and timing of diagnosis were captured. Data were assessed for all countries combined (global) and within each country and physician specialty. RESULTS: A total of 3346 PRFs were submitted. Approximately half of patients received diagnosis within 6 months. There were large country differences. In France, only 35% of patients were diagnosed within 6 months compared to 65% in Japan. Physicians in France also reported diagnoses taking > 9 months for a substantial number of patients (39%) compared with other countries (16-29%). Caregivers were the main driver toward diagnosis. Physician suspicion of AD was a trigger for diagnosis in only 20% of cases, globally. Overall, referral rates were low (14-23%). CONCLUSION: This study suggests that detection and timely diagnosis of AD remains suboptimal. This highlights the importance of fostering awareness of early symptoms and education on the benefits of timely diagnosis, a critical step in initiating treatment as early as possible.

Alzheimer's Treatment: Real-World Physician Behavior Across Countries.

OBJECTIVE: Timely initiation of Alzheimer's disease (AD)-specific treatment may postpone cognitive deterioration and preserve patient independence. We explored real-world physician behavior in the treatment of AD. METHODS: Online questionnaires and patient record forms (PRFs) were completed by participating physicians. The physicians included general practitioners, neurologists, geriatricians and psychiatrists, recruited from France, Germany, Japan, the UK and the USA. Physicians completed an online interview and two to three PRFs based on selected records of their patients with AD. Data on treatment algorithms and key drivers for therapy were captured. RESULTS: A total of 3346 PRFs were submitted and 1086 physicians interviewed. Overall, 44% of patients with mild cognitive impairment/prodromal AD, 71% of patients with mild disease and 76% of patients with moderate disease had already received therapy. The most common reasons for not prescribing therapy were patient refusal (35%) and early disease stage (26%). Except in the USA, the majority of physicians preferred to prescribe monotherapy. Almost 30% of patients at any stage of the disease did not receive AD-specific pharmacotherapy immediately after diagnosis. CONCLUSIONS: Physicians' attitudes toward AD treatment could be driven by limited awareness regarding the benefits of early intervention and the modest efficacy of currently available therapies. Efficacious therapies for AD, especially early AD, which could be used alone or in combination with current medications to maximize treatment benefit, are still needed. The availability of more efficacious therapies may improve time to treatment initiation, treatment rates and acceptance of treatment by patients, caregivers and physicians.

Insulin and IGF-2 support rat corneal endothelial cell growth and wound repair in the organ cultured tissue.

The ability of insulin and IGF-2 to support wound repair in the organ-cultured rat corneal endothelium was investigated. Corneas given a circular transcorneal freeze injury, were explanted into organ cultures containing either insulin or IGF-2 and cultured up to72 h. Both factors increased [3H]-thymidine incorporation and mitotic levels compared to controls. Insulin's ability to mediate wound closure without serum was dependent on its continuous presence in the medium. PKC was also investigated in endothelial repair using the PKC promoter phorbol 12-myristate 13-acetate (PMA). Concentrations between 10-6 and 10-8 M, PMA failed to accelerate wound closure. When injured endothelia were cultured in the presence of insulin and the PKC inhibitor H-7, wound closure was also unaffected. These results indicate that insulin and IGF-2 stimulate cell growth in injured rat corneal endothelium and that insulin without the benefit of serum promotes wound closure in situ independent of the PKC pathway.

Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision Using a Zigzag Force Ramp.

Precise quantification of the energetics and interactions that stabilize membrane proteins in a lipid bilayer is a long-sought goal. Toward this end, atomic force microscopy has been used to unfold individual membrane proteins embedded in their native lipid bilayer, typically by retracting the cantilever at a constant velocity. Recently, unfolding intermediates separated by as few as two amino acids were detected using focused-ion-beam-modified ultrashort cantilevers. However, unambiguously discriminating between such closely spaced states remains challenging, in part because any individual unfolding trajectory only occupies a subset of the total number of intermediates. Moreover, structural assignment of these intermediates via worm-like-chain analysis is hindered by brief dwell times compounded with thermal and instrumental noise. To overcome these issues, we moved the cantilever in a sawtooth pattern of 6-12 nm, offset by 0.25-1 nm per cycle, generating a "zigzag" force ramp of alternating positive and negative loading rates. We applied this protocol to the model membrane protein bacteriorhodopsin (bR). In contrast to conventional studies that extract bR's photoactive retinal along with the first transmembrane helix, we unfolded bR in the presence of its retinal. To do so, we introduced a previously developed enzymatic-cleavage site between helices E and F and pulled from the top of the E helix using a site-specific, covalent attachment. The resulting zigzag unfolding trajectories occupied 40% more states per trajectory and occupied those states for longer times than traditional constant-velocity records. In total, we identified 31 intermediates during the unfolding of five helices of EF-cleaved bR. These included a previously reported, mechanically robust intermediate located between helices C and B that, with our enhanced resolution, is now shown to be two distinct states separated by three amino acids. Interestingly, another intermediate directly interacted with the retinal, an interaction confirmed by removing the retinal.

Quantitative analysis tool for clinical functional MRI in mild traumatic brain injury.

Functional magnetic resonance imaging (fMRI) has been available commercially for clinical diagnostic use for many years. However, both clinical interpretation of fMRI by a neuroradiologist and quantitative analysis of fMRI data can require significant personnel resources that exceed reimbursement. In this report, a fully automated computer-based quantification methodology (Enumerated Auditory Response, EAR) has been developed to provide an auditory fMRI assessment of patients who have suffered a mild traumatic brain injury. Fifty-five study participants with interpretable auditory fMRI sequence data were assessed by EAR analysis, as well as both clinical radiologist fMRI interpretation and voxelwise general linear model (GLM) analysis. Comparison between the clinical interpretation and the two computer analysis methods resulted in 67% concordance (identical), 32% nearconcordance (one level difference), and 1% discordant. Comparison between the clinical computer-based quantification (EAR) and GLM analysis yielded significant correlations in right and left ear responses (p⟨0.05) for the full subject group. Automated fMRI quantification analysis equivalent to EAR might be appropriate for both future research projects with constrained resources, as well as possible routine clinical use.

Analysis of magnetic resonance spectroscopy relative metabolite ratios in mild traumatic brain injury and normative controls.

INTRODUCTION: We evaluated magnetic resonance spectroscopy (MRS) in United States military personnel with persistent symptoms after mild traumatic brain injury (mTBI), comparing over time two groups randomized to receive hyperbaric oxygen or sham chamber sessions and a third group of normative controls. METHODS: Active-duty or veteran military personnel and normative controls underwent MRS outcome measures at baseline, 13 weeks (mTBI group only), and six months. Participants received 3.0 Tesla brain MRS for analysis of water-suppressed two-dimensional (2D) multivoxel 1H-MRS of the brain using point resolved spectroscopy (PRESS) with volume selection localized above the lateral ventricles and within the brain parenchyma, of which one voxel was chosen in each hemisphere without artifact. Script-based automatic data processing was used to assess N-acetylaspartate (NAA), creatine (Cr), and choline (Cho). Metabolite ratios for white matter were then calculated for NAA/Cr (Area), Cho/Cr (Area), and Cho/NAA (Area). These ratios were compared using standard analysis methodology. RESULTS: There were no observable differences between participants with mTBI and normative controls nor any observable changes over time in the NAA/Cr (area), Cho/Cr (area), and Cho/NAA (area) ratios. Similarly, the control and injured participants were indistinguishable. DISCUSSION: While participants with mild TBI showed no difference in MRS compared to normative controls, our results are limited by the few voxels chosen and potentially by less sensitive MRS markers.

Imaging DNA Equilibrated onto Mica in Liquid Using Biochemically Relevant Deposition Conditions.

For over 25 years, imaging of DNA by atomic force microscopy has been intensely pursued. Ideally, such images are then used to probe the physical properties of DNA and characterize protein-DNA interactions. The atomic flatness of mica makes it the preferred substrate for high signal-to-noise ratio (SNR) imaging, but the negative charge of mica and DNA hinders deposition. Traditional methods for imaging DNA and protein-DNA complexes in liquid have drawbacks: DNA conformations with an anomalous persistence length ( p), low SNR, and/or ionic deposition conditions detrimental to preserving protein-DNA interactions. Here, we developed a process to bind DNA to mica in a buffer containing both MgCl2 and KCl that resulted in high SNR images of equilibrated DNA in liquid. Achieving an equilibrated 2D configuration ( i. e., p = 50 nm) not only implied a minimally perturbative binding process but also improved data quality and quantity because the DNA's configuration was more extended. In comparison to a purely NiCl2-based protocol, we showed that an 8-fold larger fraction (90%) of 680-nm-long DNA molecules could be quantified. High-resolution images of select equilibrated molecules revealed the right-handed structure of DNA with a helical pitch of 3.5 nm. Deposition and imaging of DNA was achieved over a wide range of monovalent and divalent ionic conditions, including a buffer containing 50 mM KCl and 3 mM MgCl2. Finally, we imaged two protein-DNA complexes using this protocol: a restriction enzyme bound to DNA and a small three-nucleosome array. We expect such deposition of protein-DNA complexes at biochemically relevant ionic conditions will facilitate biophysical insights derived from imaging diverse protein-DNA complexes.

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.

High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy.

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.

FEATHER: Automated Analysis of Force Spectroscopy Unbinding and Unfolding Data via a Bayesian Algorithm.

Single-molecule force spectroscopy (SMFS) provides a powerful tool to explore the dynamics and energetics of individual proteins, protein-ligand interactions, and nucleic acid structures. In the canonical assay, a force probe is retracted at constant velocity to induce a mechanical unfolding/unbinding event. Next, two energy landscape parameters, the zero-force dissociation rate constant (ko) and the distance to the transition state (Δx‡), are deduced by analyzing the most probable rupture force as a function of the loading rate, the rate of change in force. Analyzing the shape of the rupture force distribution reveals additional biophysical information, such as the height of the energy barrier (ΔG‡). Accurately quantifying such distributions requires high-precision characterization of the unfolding events and significantly larger data sets. Yet, identifying events in SMFS data is often done in a manual or semiautomated manner and is obscured by the presence of noise. Here, we introduce, to our knowledge, a new algorithm, FEATHER (force extension analysis using a testable hypothesis for event recognition), to automatically identify the locations of unfolding/unbinding events in SMFS records and thereby deduce the corresponding rupture force and loading rate. FEATHER requires no knowledge of the system under study, does not bias data interpretation toward the dominant behavior of the data, and has two easy-to-interpret, user-defined parameters. Moreover, it is a linear algorithm, so it scales well for large data sets. When analyzing a data set from a polyprotein containing both mechanically labile and robust domains, FEATHER featured a 30-fold improvement in event location precision, an eightfold improvement in a measure of the accuracy of the loading rate and rupture force distributions, and a threefold reduction of false positives in comparison to two representative reference algorithms. We anticipate FEATHER being leveraged in more complex analysis schemes, such as the segmentation of complex force-extension curves for fitting to worm-like chain models and extended in future work to data sets containing both unfolding and refolding transitions.