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1.
Cell ; 170(3): 577-592.e10, 2017 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-28753431

RESUMEN

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.


Asunto(s)
Neoplasias/genética , Neoplasias/patología , Interferencia de ARN , Línea Celular Tumoral , Biblioteca de Genes , Redes Reguladoras de Genes , Humanos , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oncogenes , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción/metabolismo
2.
Proc Natl Acad Sci U S A ; 121(7): e2313818121, 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38324569

RESUMEN

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.


Asunto(s)
Bacteriorodopsinas , Bacteriorodopsinas/metabolismo , Ligandos , Análisis Espectral , Retina/metabolismo , Conformación Molecular , Conformación Proteica
3.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33753487

RESUMEN

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.


Asunto(s)
Bacteriorodopsinas/química , Pliegue de Proteína , Termodinámica , Sustitución de Aminoácidos , Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Halobacterium salinarum , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza Atómica , Mutación Puntual , Imagen Individual de Molécula
4.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33723041

RESUMEN

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.


Asunto(s)
Microscopía de Fuerza Atómica/métodos , Modelos Moleculares , Pliegue de Proteína , Proteínas/química , Concentración de Iones de Hidrógeno , Fenómenos Mecánicos , Microscopía de Fuerza Atómica/instrumentación , Imagen Individual de Molécula
5.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33723050

RESUMEN

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.


Asunto(s)
Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Proteínas Bacterianas/química , Bacterias Gramnegativas/fisiología , Bacterias Gramnegativas/ultraestructura , Microscopía de Fuerza Atómica , Estabilidad Proteica , Salmonella/fisiología , Salmonella/ultraestructura , Termodinámica
6.
Nucleic Acids Res ; 48(6): 2969-2981, 2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-32043141

RESUMEN

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.


Asunto(s)
ADN/química , Imagenología Tridimensional , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , Complejo Represivo Polycomb 2/metabolismo , Humanos , Unión Proteica , Multimerización de Proteína
7.
Biophys J ; 118(3): 667-675, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-31882249

RESUMEN

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.


Asunto(s)
Bacteriorodopsinas , Desplegamiento Proteico , Bacteriorodopsinas/metabolismo , Membrana Dobles de Lípidos , Microscopía de Fuerza Atómica , Desnaturalización Proteica , Pliegue de Proteína , Retina
8.
Growth Factors ; 38(5-6): 269-281, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-34388064

RESUMEN

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.


Asunto(s)
Factor II del Crecimiento Similar a la Insulina , Insulina , Animales , Células Cultivadas , Células Endoteliales , Endotelio Corneal , Factor I del Crecimiento Similar a la Insulina , Técnicas de Cultivo de Órganos , Ratas
9.
Phys Rev Lett ; 125(6): 068102, 2020 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-32845671

RESUMEN

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.


Asunto(s)
Bacteriorodopsinas/química , Modelos Químicos , Microscopía de Fuerza Atómica , Pliegue de Proteína , Termodinámica
10.
Nucleic Acids Res ; 45(18): 10775-10782, 2017 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-28977580

RESUMEN

Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein-DNA interactions.


Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Unión Proteica , RecQ Helicasas/metabolismo
11.
Nano Lett ; 18(10): 6318-6325, 2018 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-30234311

RESUMEN

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.


Asunto(s)
VIH/ultraestructura , Nanotecnología , Conformación de Ácido Nucleico , ARN Viral/ultraestructura , VIH/química , Humanos , Microscopía de Fuerza Atómica , Pinzas Ópticas , ARN Viral/química , Imagen Individual de Molécula
12.
Undersea Hyperb Med ; 46(3): 291-297, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31394599

RESUMEN

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.


Asunto(s)
Ácido Aspártico/análogos & derivados , Química Encefálica , Conmoción Encefálica/metabolismo , Colina/análisis , Creatina/análisis , Espectroscopía de Resonancia Magnética/métodos , Adulto , Ácido Aspártico/análisis , Conmoción Encefálica/terapia , Estudios de Casos y Controles , Femenino , Humanos , Oxigenoterapia Hiperbárica , Ventrículos Laterales/química , Masculino , Personal Militar , Síndrome Posconmocional/metabolismo , Factores de Tiempo , Veteranos
13.
Undersea Hyperb Med ; 46(3): 245-249, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31394595

RESUMEN

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.


Asunto(s)
Enfermedades Auditivas Centrales/diagnóstico por imagen , Conmoción Encefálica/fisiopatología , Diagnóstico por Computador/métodos , Técnicas de Diagnóstico Otológico , Imagen por Resonancia Magnética/métodos , Enfermedades Auditivas Centrales/fisiopatología , Conmoción Encefálica/diagnóstico por imagen , Femenino , Humanos , Modelos Lineales , Masculino , Personal Militar , Veteranos
14.
Angew Chem Int Ed Engl ; 58(6): 1710-1713, 2019 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-30556941

RESUMEN

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.


Asunto(s)
Bacteriorodopsinas/química , Retina/química , Bacteriorodopsinas/metabolismo , Modelos Moleculares , Desplegamiento Proteico , Retina/metabolismo
15.
Biophys J ; 115(5): 757-762, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-30122292

RESUMEN

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.


Asunto(s)
Algoritmos , Desplegamiento Proteico , Análisis Espectral , Automatización , Teorema de Bayes , Termodinámica
16.
Chemphyschem ; 19(1): 19-23, 2018 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-29069529

RESUMEN

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.


Asunto(s)
Diseño Asistido por Computadora , Digoxigenina/química , Proteínas/química , Termodinámica , Sitios de Unión , Ligandos , Microscopía de Fuerza Atómica
17.
J Chem Phys ; 148(12): 123313, 2018 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-29604885

RESUMEN

Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force microscopy (AFM) emerged as a powerful tool to mechanically extract individual membrane proteins from a lipid bilayer. Concurrently, fluctuation theorems, such as the Jarzynski equality, were applied to deduce equilibrium free energies (ΔG0) from non-equilibrium single-molecule force spectroscopy records. The combination of these two advances in single-molecule studies deduced the free-energy of the model membrane protein bacteriorhodopsin in its native lipid bilayer. To elucidate this free-energy landscape at a higher resolution, we applied two recent developments. First, as an input to the reconstruction, we used force-extension curves acquired with a 100-fold higher time resolution and 10-fold higher force precision than traditional AFM studies of membrane proteins. Next, by using an inverse Weierstrass transform and the Jarzynski equality, we removed the free energy associated with the force probe and determined the molecular free-energy landscape of the molecule under study, bacteriorhodopsin. The resulting landscape yielded an average unfolding free energy per amino acid (aa) of 1.0 ± 0.1 kcal/mol, in agreement with past single-molecule studies. Moreover, on a smaller spatial scale, this high-resolution landscape also agreed with an equilibrium measurement of a particular three-aa transition in bacteriorhodopsin that yielded 2.7 kcal/mol/aa, an unexpectedly high value. Hence, while average unfolding ΔG0 per aa is a useful metric, the derived high-resolution landscape details significant local variation from the mean. More generally, we demonstrated that, as anticipated, the inverse Weierstrass transform is an efficient means to reconstruct free-energy landscapes from AFM data.


Asunto(s)
Bacteriorodopsinas/química , Termodinámica , Membrana Dobles de Lípidos/química , Microscopía de Fuerza Atómica , Pliegue de Proteína
18.
Nucleic Acids Res ; 44(12): 5849-60, 2016 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-27220465

RESUMEN

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 µM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.


Asunto(s)
ADN Bacteriano/química , ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Exodesoxirribonucleasa V/química , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Sitios de Unión , ADN/genética , ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasa V/genética , Exodesoxirribonucleasa V/metabolismo , Expresión Génica , Cinética , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica
19.
Biophys J ; 113(12): 2595-2600, 2017 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-29132641

RESUMEN

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.


Asunto(s)
Microscopía de Fuerza Atómica/instrumentación , Diseño de Equipo , Oro
20.
J Struct Biol ; 197(1): 13-25, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-26804584

RESUMEN

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.


Asunto(s)
Microscopía de Fuerza Atómica/métodos , Proteínas/química , Imagen Individual de Molécula/métodos , Biofisica , Oro , Nanotecnología/métodos , Pliegue de Proteína
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