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1.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2213-2216, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018447

RESUMO

The Refractive Index (RI) is an important parameter of characterizing optical properties of particles. In a dual-beam optical trap, two counter-propagating laser beams are used to trap micro-particles suspended in an aqueous medium. When a ray of light passes from one medium of lower RI (e.g. aqueous suspension medium) to another medium of higher RI (e.g. suspended particle), its momentum changes which exerts a proportional trapping force on the surface of the particle. Thus, accurate knowledge of RI of the particles and the surrounding medium is needed to determine the behavior of particles in an optical trap. The RI of micro-sized beads can be experimentally measured using traditional optical methods such as absorption microscopy. We developed an alternative theoretical method to estimate the RI of trapped particles based on non-contact optical trapping experimental outcomes. In our study, a theoretical model was formulated based on the experimentally measured minimum trapping powers for polystyrene and polyethylene beads using a dual-beam optical setup. The tendencies of trapping power-RI curves predicted by our model agreed very well with those measured experimentally. Our technique provides an alternative approach to determining the RI of a certain micro-size particle regardless of its size or density. Our method is especially advantageous over traditional methods to determine RI of biological particles which exhibit significant variations based on physiological and environmental conditions.


Assuntos
Pinças Ópticas , Refratometria , Microscopia , Modelos Teóricos , Poliestirenos
2.
Nat Commun ; 11(1): 4714, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948754

RESUMO

The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.


Assuntos
DNA/química , Fenômenos Magnéticos , Magnetismo/métodos , Nanotecnologia , Análise Espectral/métodos , Ciprofloxacino/farmacologia , DNA/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Conformação de Ácido Nucleico , Pinças Ópticas , Fenômenos Físicos , Software
3.
Proc Natl Acad Sci U S A ; 117(27): 15650-15658, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571937

RESUMO

Liquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Lipídeos/química , Nucleoproteínas/química , RNA/química , Anisotropia , Proteínas Intrinsicamente Desordenadas/genética , Lipídeos/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Nucleoproteínas/genética , Pinças Ópticas , Transição de Fase , RNA/genética
4.
Nat Commun ; 11(1): 2060, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345978

RESUMO

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.


Assuntos
Conectina/metabolismo , Endopeptidases/genética , Especificidade de Órgãos , Animais , Fenômenos Biomecânicos , Conectina/química , Feminino , Proteínas Imobilizadas/metabolismo , Magnetismo , Camundongos , Músculos/metabolismo , Músculos/ultraestrutura , Pinças Ópticas , Fenótipo , Dobramento de Proteína , Análise Espectral
5.
Proc Natl Acad Sci U S A ; 117(17): 9318-9328, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32273391

RESUMO

Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.


Assuntos
Adenosina Trifosfatases/metabolismo , Alquil e Aril Transferases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Alquilação/fisiologia , DNA/metabolismo , Dano ao DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Guanina/metabolismo , Microscopia de Força Atômica/métodos , Mutagênese , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinças Ópticas
6.
Nucleic Acids Res ; 48(9): 5024-5036, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32282908

RESUMO

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.


Assuntos
Sequência Rica em At , DNA de Helmintos/química , Animais , Fenômenos Biomecânicos , Caenorhabditis elegans/genética , DNA de Helmintos/ultraestrutura , Genoma Helmíntico , Microscopia de Força Atômica , Pinças Ópticas
7.
Phys Rev Lett ; 124(5): 058101, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32083903

RESUMO

We report that trivalent cobalt hexammine cations decrease the persistence length, stretching modulus, helical density, and size of plectonemes formed under torque of DNA but increase those of RNA. Divalent magnesium cations, however, decrease the persistence lengths, contour lengths, and sizes of plectonemes while increasing the helical densities of both DNA and RNA. The experimental results are explained by different binding modes of the cations on DNA and RNA in our all-atom molecular dynamics simulations. The significant variations of the helical densities and structures of DNA and RNA duplexes induced by high-valent cations may affect interactions of the duplexes with proteins.


Assuntos
DNA/química , RNA de Cadeia Dupla/química , Cátions/química , Cobalto/química , Elasticidade , Magnésio/química , Magnetismo/instrumentação , Magnetismo/métodos , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Pinças Ópticas , RNA/química
8.
J Chem Phys ; 152(7): 074204, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32087630

RESUMO

Identification of defective DNA structures is a difficult task, since small differences in base-pair bonding are hidden in the local structural variability of a generally random base-pair sequence. Defects, such as base mismatches, missing bases, crosslinks, and so on, occur in DNA with high frequency and must be efficiently identified and repaired to avoid dire consequences such as genetic mutations. Here, we focus on the detection of base mismatches, which is local deviations from the ideal Watson-Crick pairing rule, which may typically originate from DNA replication process, foreign chemical attack, or ionizing radiation. Experimental detection of a mismatch defect demands the ability to measure slight deviations in the free energy and molecular structure. We introduce different mismatches in short DNA hairpins (10 or 20 base pairs plus a 4-base loop) sandwiched between dsDNA handles to be used in single-molecule force spectroscopy with optical tweezers. We perform both hopping and force-pulling experiments to measure the excess free energies and deduce the characteristic kinetic signatures of the mismatch from the force-distance curves. All-atom molecular dynamics simulations lend support to the detailed interpretation of the experimental data. Such measurements, at the lowest sensitivity limits of this experimental technique, demonstrate the capability of identifying the presence of mismatches in a random complementary dsDNA sequence and provide lower bounds for the ability to distinguish different structural defects.


Assuntos
Simulação de Dinâmica Molecular , Pareamento Incorreto de Bases , DNA/química , Microscopia de Força Atômica , Conformação de Ácido Nucleico , Pinças Ópticas
9.
Nat Chem Biol ; 16(7): 801-809, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32066968

RESUMO

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.


Assuntos
DNA/metabolismo , Quadruplex G , RNA/metabolismo , Telomerase/metabolismo , Telômero/enzimologia , Biocatálise , DNA/genética , Replicação do DNA , Expressão Gênica , Células HEK293 , Humanos , Pinças Ópticas , RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Telomerase/genética , Telômero/ultraestrutura
10.
Chem Commun (Camb) ; 56(14): 2123-2126, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-31970368

RESUMO

Here, we develop an optical tweezers-based single-molecule manipulation assay to detect the formation of an R-loop complex in the Cas12a system and characterize its thermodynamic stability. We found that the formation of the R-loop complex induces a two-step unfolding of a DNA hairpin containing the target sequence, the non-target sequence binds loosely to Cas12a and can be easily released from the complex, and the Nuc domain of Cas12a plays key roles in target binding and R-loop formation.


Assuntos
DNA/síntese química , Sistemas CRISPR-Cas/genética , DNA/química , DNA/genética , Pinças Ópticas , Termodinâmica
11.
Nature ; 578(7794): 317-320, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996849

RESUMO

The ability to reverse protein aggregation is vital to cells1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp704,5. However, the model is also controversial, as the necessary motor activity has not been identified6-8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore11,12. Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)13 and degradation by the 26S proteasome14.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Agregados Proteicos , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Fluorescência , Proteínas de Choque Térmico/química , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Pinças Ópticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Redobramento de Proteína , Ubiquitina/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(2): 1036-1041, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888993

RESUMO

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.


Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Exocitose/fisiologia , Cinética , Fusão de Membrana/fisiologia , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
13.
Development ; 147(3)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964776

RESUMO

Directional cell intercalations of epithelial cells during gastrulation has, in several organisms, been shown to be associated with a planar cell polarity in the organisation of the actin-myosin cytoskeleton and is postulated to reflect directional tension that drives oriented cell intercalations. We have characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual epithelial cell junctions of cells in various locations and orientations in the epiblast of chick embryos in the early stages of primitive streak formation. Junctional tension of mesendoderm precursors in the epiblast is higher in junctions oriented in the direction of intercalation than in junctions oriented perpendicular to the direction of intercalation and higher than in junctions of other cells in the epiblast. The kinetic data fit best with a simple viscoelastic Maxwell model, and we find that junctional tension, and to a lesser extent viscoelastic relaxation time, are dependent on myosin activity.


Assuntos
Células Epiteliais/metabolismo , Gastrulação/fisiologia , Junções Intercelulares/metabolismo , Pinças Ópticas , Linha Primitiva/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Embrião de Galinha , Gástrula/metabolismo , Camadas Germinativas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrocarbonetos Clorados/farmacologia , Microscopia de Fluorescência/métodos , Miosina Tipo I/antagonistas & inibidores , Miosina Tipo I/metabolismo , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/metabolismo , Pirróis/farmacologia , Transdução de Sinais/fisiologia
14.
PLoS One ; 14(12): e0225773, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805109

RESUMO

We present numerical methods for modeling the dynamics of arbitrarily shaped particles trapped within optical tweezers, which improve the predictive power of numerical simulations for practical use. We study the dependence of trapping on the shape and size of particles in a single continuous wave beam setup. We also consider the implications of different particle compositions, beam types and media. The major result of the study is that for different irregular particle shapes, a range of beam powers generally leads to trapping. The trapping power range depends on whether the particle can be characterized as elongated or flattened, and the range is also limited by Brownian forces.


Assuntos
Análise Numérica Assistida por Computador , Pinças Ópticas , Simulação por Computador , Campos Eletromagnéticos , Movimento (Física) , Distribuição Normal , Rotação
15.
PLoS One ; 14(10): e0223688, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31671114

RESUMO

Insights into the morphology of nanoclusters would facilitate the design of nano-devices with improved optical, electrical, and magnetic responses. We have utilized optical gradient forces for the directed self-assembly of colloidal clusters using high-repetition-rate femtosecond laser pulses to delineate their structure and dynamics. We have ratified our experiments with theoretical models derived from the Langevin equation and defined the valid ranges of applicability. Our femtosecond optical tweezer-based technique characterizes the in-situ formation of hierarchical self-assembled clusters of homomers as well as heteromers by analyzing the back focal plane displacement signal. This technique is able to efficiently distinguish between nano-particles in heterogeneous clusters and is in accordance with our theory. Herein, we report results from our technique, and also develop a model to describe the mechanism of such processes where corner frequency changes. We show how the corner frequency changes enables us to recognize the structure and dynamics of the coagulation of colloidal homogeneous and heterogeneous clusters in condensed media over a broad range of nanoparticle sizes. The methods described here are advantageous, as the backscatter position-sensitive detection probes the in-situ self-assembly process while other light scattering approaches are leveraged for the characterization of isolated clusters.


Assuntos
Modelos Teóricos , Nanoestruturas , Pinças Ópticas , Algoritmos
16.
Phys Rev Lett ; 123(18): 188102, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31763918

RESUMO

The cytoskeleton is a composite network of three types of protein filaments, among which intermediate filaments (IFs) are the most extensible ones. Two very important IFs are keratin and vimentin, which have similar molecular architectures but different mechanical behaviors. Here we compare the mechanical response of single keratin and vimentin filaments using optical tweezers. We show that the mechanics of vimentin strongly depends on the ionic strength of the buffer and that its force-strain curve suggests a high degree of cooperativity between subunits. Indeed, a computational model indicates that in contrast to keratin, vimentin is characterized by strong lateral subunit coupling of its charged monomers during unfolding of α helices. We conclude that cells can tune their mechanics by differential use of keratin versus vimentin.


Assuntos
Citoesqueleto/química , Queratinas/química , Modelos Biológicos , Vimentina/química , Fenômenos Biomecânicos , Tampões (Química) , Citoesqueleto/metabolismo , Queratinas/metabolismo , Microscopia de Força Atômica , Pinças Ópticas , Concentração Osmolar , Conformação Proteica em alfa-Hélice , Vimentina/metabolismo
17.
Nat Commun ; 10(1): 5321, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757965

RESUMO

Surface-enhanced Raman spectroscopy (SERS) sensing of DNA bases by plasmonic nanopores could pave a way to novel methods for DNA analyses and new generation single-molecule sequencing platforms. The SERS discrimination of single DNA bases depends critically on the time that a DNA strand resides within the plasmonic hot spot. In fact, DNA molecules flow through the nanopores so rapidly that the SERS signals collected are not sufficient for single-molecule analysis. Here, we report an approach to control the residence time of molecules in the hot spot by an electro-plasmonic trapping effect. By directly adsorbing molecules onto a gold nanoparticle and then trapping the single nanoparticle in a plasmonic nanohole up to several minutes, we demonstrate single-molecule SERS detection of all four DNA bases as well as discrimination of single nucleobases in a single oligonucleotide. Our method can be extended easily to label-free sensing of single-molecule amino acids and proteins.


Assuntos
DNA/análise , Nanopartículas Metálicas , Nanoporos , Pinças Ópticas , Imagem Individual de Molécula/métodos , Análise Espectral Raman/métodos , Adenina/análise , Citosina/análise , DNA/química , Ouro , Guanina/análise , Óptica e Fotônica , Timina/análise
18.
Proc Natl Acad Sci U S A ; 116(51): 25641-25648, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776255

RESUMO

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.


Assuntos
Biossíntese de Proteínas/fisiologia , Domínios Proteicos/fisiologia , Dobramento de Proteína , Proteínas , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Pinças Ópticas , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Ribossomos , Imagem Individual de Molécula , Termodinâmica
19.
PLoS One ; 14(11): e0222468, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31697710

RESUMO

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single-molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1-aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer-Mg+2 complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Saxitoxina/química , Simulação de Dinâmica Molecular , Nanotecnologia/métodos , Neurotoxinas/química , Pinças Ópticas
20.
Phys Chem Chem Phys ; 21(44): 24527-24534, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31663550

RESUMO

Folding is generally assumed to be a Markov process, without memory. When the molecular motion is coupled to that of a probe as in single-molecule force spectroscopy (SMFS) experiments, however, theory predicts that the coupling to a second Markov process should induce memory when monitoring a projection of the full multi-dimensional motion onto a reduced coordinate. We developed a method to evaluate the time constant of the induced memory from its effects on the autocorrelation function, which can be readily determined from experimental data. Applying this method to both simulated SMFS measurements and experimental trajectories of DNA hairpin folding measured by optical tweezers as a model system, we validated the prediction that the linker induces memory. For these measurements, the timescale of the induced memory was found to be similar to the time required for the force probe to respond to changes in the molecule, and in the regime where the experimentally observed dynamics were not significantly perturbed by probe-molecule coupling artifacts. Memory effects are thus a general feature of SMFS measurements induced by the mechanical connection between the molecule and force probe that should be considered when interpreting experimental data.


Assuntos
DNA/química , Imagem Individual de Molécula , DNA/metabolismo , Sequências Repetidas Invertidas , Cinética , Cadeias de Markov , Conformação de Ácido Nucleico , Pinças Ópticas
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