28 research outputs found
A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues
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
The Mechanical Power of Titin Folding
Summary: The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band. We find that formation of the disulfide regulates the peak power output of protein folding in an all-or-none manner, providing at 6.0 pN, for example, a boost from 0 to 6,000 zW upon oxidation. This mechanism of power generation from protein folding is of great importance for muscle, where titin domains may unfold and refold with each extension and contraction of the sarcomere. : Eckels et al. use single-molecule magnetic tweezers to simultaneously probe the folding dynamics of titin Ig domains and monitor the redox status of single disulfides within the Ig fold. Oxidation of the disulfide bond greatly increases both the folding force and the magnitude of power delivered by protein folding. Keywords: protein folding, titin, single molecule, magnetic tweezers, force spectroscopy, disulfide bond, mechanical power, muscle contraction, oxidative folding, oxidoreductas
Work Done by Titin Protein Folding Assists Muscle Contraction
Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin immunoglobulin (Ig) domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6–8 pN. We use single-molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but as yet unrecognized, contributor to the force generated by a contracting muscle
A FORMAÇÃO DE PROFESSORES PARA A EDUCAÇÃO EM DIREITOS HUMANOS ANALISADA SOB A ÓTICA DA ORGANIZAÇÃO DO TRABALHO DIDÁTICO: TECENDO ALGUMAS CONSIDERAÇÕES
Este trabalho propõe-se a traçar uma breve discussão sobre a formação de professores para a educação em direitos humanos sob a ótica da organização do trabalho didático, discutindo a escola manufatureira que se consolidou no final do século XIX e início do século XX para atender às demandas do próprio capital, bem como os principais movimentos que influenciaram na organização do trabalho didático dessa escola. E claro que não poderíamos deixar de iniciar nossa discussão apresentando a apropriação do direito no século XVIII pelos filósofos iluministas, para chegarmos à concepção de Estado que temos hoje: liberal, democrático e de direito. Nossas considerações sobre as formações em educação em direitos humanos, deu-se através da análise do material didático utilizado na Capacitação de Educadores da Rede Básica em Educação em Direitos Humanos-REDH BRASIL, onde evidenciou-se que os discursos que adentram a escola hoje, embora primem pela igualdade, pela cidadania e pela co-responsabilidade social, não possibilitam a quem passa pelos seus bancos apreender os movimentos históricos e a organização da sociedade capitalista e, assim, o reconhecimento da real dignidade humana
Kinetic parameters for ADP-dependent glucokinase from <i>T. litoralis</i>.
‡<p>Values obtained from fitting the data to the linear expression of bi-substrate ordered sequential model.</p
Interactions clusters between the small and large domain that mediate the transition from the open to the closed conformation.
<p>(A) Structure of the enzyme in the apo-form and (B) structure of the ternary TlGK·Mg·ADPβS·D-glucose complex. The small domain was colored in yellow (apo-enzyme) or blue (ternary complex) for clarity purposes. Side chains of residues are represented as sticks. The atoms involved in H-bonds are represented as spheres. The nucleotide (ADP), the cosolvent, glycerol (GOL) and water (W) are also shown. Cluster 1 achieves communication between the small and large domain through Glu188, Thr446 and Val447. Cluster 2 residues contribute to stabilize the ADP-induced conformational change. In the apo-enzyme, Arg202 of the large domain is over the active-site pocket and the side chain of Tyr354 is oriented toward the outside of the pocket. In contrast, in the TlGK·Mg·ADPβS·D-glucose ternary complex Arg202 and Tyr354 form a stacking interaction afforded by the rotation of their side-chains so as to allow a cation-π interaction. Cluster 3′s interaction network is rearranged upon ADP binding through the formation of H-bonds between the large and small domain that generates a net attraction. In cluster 4, Arg191 from the small domain H-bonds Ala441 and Ser442 from the small domain. The only net repulsive interaction is provided by cluster 5, involving Lys74 and Lys246; in the apo-enzyme Glu100 is H-bonded to Lys74 thus neutralizing its net charge, but in the ternary complex this stabilizing interaction is absent and Lys74 comes closer to Lys246 despite the associated unfavorable energy barrier.</p
Scattering curves and pair distance distribution functions <i>P(r)</i>.
<p>(<b>A</b>) Scattering patterns for the different conditions explored: apoenzyme (□), enzyme-D-glucose (▪), enzyme-Mg·ADP (○) and in the presence of Mg·ADPβS and glucose (•). The curves were normalized to unity at their maximum value for comparison purposes. (<b>B</b>) P(r) graphs for each condition calculated by Fourier transformation using GNOM (28). The graphs were normalized to unity at their maximum value for comparison purposes.</p
Crystallographic data statistics and refinement.
<p>Crystallographic data statistics and refinement.</p