Glucose is a primary and essential energy source for humans. It is broken down from complex carbohydrates in the diet and absorbed across the gut epithelium into the blood stream. Glucose homeostasis is important as hyperglycermia causes damage of pancreatic and peripheral cells. In response to a meal glucose is principally taken up by fat and muscle tissues and this response is activated by insulin release from pancreatic beta cells. Insulin stimulates the translocation of GLUT4 from the intracellular storage vesicles to the plasma membrane in fat and muscle cells. Although many proteins have been implicated in this process, the key insulin-regulated substrate has not been determined yet. In the present study, the phosphoserine/threonine binding protein 14-3-3 was used as a tool to affinity-purify insulin-stimulated phosphoproteins from 3T3-L1 adipocytes. By using mass spectrometry 38 proteins were identified, reflecting the important role of 14-3-3 in mediating many insulin-regulated processes. Among the potential phosphoproteins was Myosin 1C (Myo1c), an actin-associated molecular motor, which has previously been implicated in insulin-stimulated GLUT4 trafficking in adipocytes. I showed that insulin stimulates the activation of CaMKII which phosphorylates Myo1c at S701 in a Ca2+/PI3K-dependent manner. Myo1c phosphorylation induced its interaction with 14-3-3-proteins, reduced calmodulin-binding and stimulated its in vitro ATPase activity. Insulin-dependent stimulation of Myo1c phosphorylation and its ATPase activity were both required for GLUT4 translocation. By using yeast two-hybrid techniques, I identified a candidate ligand of the Myo1c tail, Armcx5, and demonstrated the in vivo interaction in 3T3-L1 adipocytes. The siRNA-mediated knockdown of Armcx5 inhibited insulin-stimulated glucose uptake and GLUT4 translocation. These results suggest that the regulation of Myo1c and its ligand Armcx5 are essential in insulin-regulated GLUT4 trafficking, possibly playing a key role in vesicle fusion.

Diseases of heart muscle, such as hypertrophic and dilated cardiomyopathy, are often caused by mutations in proteins of the sarcomere, including actin, troponin, tropomyosin, and myosin. The molecular mechanisms of disease-causing mutations remain unclear because the process of cardiac muscle contraction and the corresponding mutational insults are incompletely defined. To elucidate the underlying mechanisms of cardiac muscle contraction, its regulation, and the effects of disease-causing mutations, the structures of sarcomeric protein assemblies must first be solved. In this dissertation we use interdisciplinary structural biology techniques, including cryo-electron microscopy (cryo-EM), protein-protein docking, and molecular dynamics simulations to investigate the interactions between actin, tropomyosin, and myosin. This structural work is foundational in identifying the molecular effects of mutations. In the first project, we present a novel cryo-EM structure of the cardiac-isoform actomyosin-tropomyosin complex. This structure, which utilizes bovine masseter β-myosin, provides the foundation for understanding the molecular effects of cardiomyopathy-causing mutations that occur at the actomyosin interface. Furthermore, by pairing our structure with protein-protein docking methods and molecular dynamics simulations, we identify complementary and periodic electrostatic interactions between the myosin surface loop 4 and tropomyosin. We hypothesize that these interactions are essential in switching between contraction- and relaxation-mediating states. In a follow up study, we test our myosin loop 4 hypotheses by creating a human cardiac β-myosin all-glycine loop 4 mutant, which abolishes nearly all electrostatic interactions between myosin and tropomyosin. After designing the mutant, we solve the cryo-EM structures of the wild-type and mutant actin-myosin-tropomyosin complexes to high resolution. Our structures confirm that the loop 4 mutant abolishes its interaction with tropomyosin and suggests that the tropomyosin cable on mutant actomyosin filaments is shifted to a new position. Subsequent molecular dynamics simulations corroborate our cryo-EM finding that tropomyosin on mutant actomyosin is displaced from the wild type position. Interaction energy calculations derived from these simulations suggest that the mutant position is significantly less stable than the wild-type. This work provides further evidence that loop 4 interactions are key in stabilizing tropomyosin position during contraction. Finally, to extend our work on the human cardiac actomyosin-tropomyosin complex, we provide insights into the ADP release step of the cardiac β-myosin kinetic cycle. Here, we use a composite method of helical and single-particle cryo-EM reconstruction techniques to solve the structures of the human cardiac actin-myosin-tropomyosin filament in the presence and absence of ADP-Mg2+. This work elucidates the structural basis of cardiac β-myosin ADP release and provides insight into the force-sensing mechanism of the cardiac motor. Lastly, we use our structures to probe how cardiomyopathy-causing mutations potentially disrupt the ADP-to-rigor transition, leading to altered myosin contractility. Overall, the structures solved in this dissertation generate fundamental understanding about the function of the cardiac thin filament and the motor protein, myosin. Moreover, this research provides a framework that connects the initial molecular insults of mutations to the disruption of proper regulation that leads to pathological progression. / 2023-08-24T00:00:00Z

Transport Molecules play a crucial role for cell viability. Amongst others, linear motors transport cargos along rope-like structures from one location of the cell to another in a stochastic fashion. Thereby each step of the motor, either forwards or backwards, bridges a fixed distance. While moving along the rope the motor can also detach and is lost. We give here a mathematical formalization of such dynamics as a random process which is an extension of Random Walks, to which we add an absorbing state to model the detachment of the motor from the rope. We derive particular properties of such processes that have not been available before. Our results include description of the maximal distance reached from the starting point and the position from which detachment takes place. Finally, we apply our theoretical results to a concrete established model of the transport molecule Kinesin V.

Rotary molecular motors are protein complexes which convert chemical or electrochemical energy from the environment into mechanical work in the form of rotary motion. The work in this thesis examines two of these motors: the F₁ portion of F₁F_O- ATP synthase, which is responsible for ATP production in bacteria and eukaryotes, and the bacterial flagellar motor (BFM), which rotates the flagella of a bacterium, enabling locomotion. The aim of these investigations was to measure the stepping dynamics of these motors, in order to further elucidate details of the stepping mechanism, the mechanism of rotation, and the mechanochemical cycle. A back-scattering laser dark field microscope of unprecedented resolution was designed and constructed to observe the rotation of gold nanoparticles attached to fixed motors. This micro- scope is capable of sub-nanometer and 20μs resolution. The protocols and algorithms to collect and analyze high resolution rotational data developed for these experiments have yielded novel discoveries for both F₁ and the BFM. While most of the previous single-molecule work has been done on F₁ from the thermophilic Bacilus PS3 (TF₁), only mitochondrial F₁ has been well characterized by high-resolution crystal structures, and single-molecule studies of mesophilic F₁ are lacking. This thesis presents evidence that mesophilic F₁ from E. coli and wild type yeast F₁ from S. cerevisiae are governed by the same mechanism as TF₁ under laboratory conditions. Experiments with yeast F₁ mutants allow a direct comparison between single-molecule rotation studies and high resolution crystal structures. A data set of unprecedented size and resolution was acquired of high speed, low load BFM rotation, enabling the first observation of steps in the BFM under physiological conditions. Preliminary results from this analysis question previously published results of the dependence of speed on stator number at low load and provide novel hypotheses necessitating new models of BFM rotation.

Motor proteins are microscopic biological machines that convert chemical energy into mechanical motion and work. They power a diverse range of biological processes, for example the swimming and crawling motion of bacteria, intracellular transport, and muscle contraction. Understanding the physical basis of these processes is interesting in its own right, but also has an interesting potential for applications in medicine and nanotechnology. The ongoing rapid developments in single molecule experimental techniques make it possible to probe these systems on the single molecule level, with increasing temporal and spatial resolution. The work presented in this thesis is concerned with physical modeling of motor proteins on the molecular scale, and with theoretical challenges in the interpretation of single molecule experiments. First, we have investigated how a small groups of elastically coupled motors collaborate, or fail to do so, when producing strong forces. Using a simple model inspired by the motor protein PilT, we find that the motors counteract each other if the density becomes higher than a certain threshold, which depends on the asymmetry of the system. Second, we have contributed to the interpretation of experiments in which the stepwise motion of a motor protein is followed in real time. Such data is naturally interpreted in terms of first passage processes. Our main conclusions are (1) Contrary to some earlier suggestions, the stepping events do not correspond to the cycle completion events associated with the work of Hill and co-workers. We have given a correct formulation. (2) Simple kinetic models predict a generic mechanism that gives rise to correlations in step directions and waiting times. Analysis of stepping data from a chimaeric flagellar motor was consistent with this prediction. (3) In the special case of a reversible motor, the chemical driving force can be extracted from statistical analysis of stepping trajectories. / QC 20100820

Amongst the many complex processes taking place in living cells, transport of cargoes across the cytosceleton is fundamental to cell viability and activity. To move cargoes between the different cell parts, cells employ Molecular Motors. The motors operate by transporting cargoes along the so-called cellular micro-tubules, namely rope-like structures that connect, for instance, the cell-nucleus and outer membrane. We introduce a new Markov Chain, the killed Quasi-Random-Walk, for such transport molecules and derive properties like the maximal run length and time. Furthermore we introduce permuted balance, which is a more flexible extension of the ordinary reversibility and introduce the notion of Time Duality, which compares certain passage times pathwise. We give a number of sufficient conditions for Time Duality based on the geometry of the transition graph. Both notions are closely related to properties of the killed Quasi-Random-Walk.

Molecular motor proteins are responsible for the long range transport of vesicles and organelles inside living cells. A small number of motor types transport thousands of distinct cargoes to various regions in the cell at the same time. This requires that intracellular transport be tightly regulated, yet the details of how motor regulators and cofactors tune motor function remain unknown in most cases. In-vitro studies at the single motor level have been instrumental in understanding the function of individual motors. In this thesis work I developed the methodology to extend in-vitro experiments to interrogate motor regulation at the single molecule level. I describe my modifications to the microscope setup as well as the acquisition cycle that made this possible. By combining differential interference contrast microscopy with single molecule fluorescence imaging and optical trapping I was able to manipulate and image the cargo while imaging a fluorescently-labeled regulator binding at the site of the motors. I used lipid droplets purified from Drosophila embryos as cargoes. Lipid droplets are carried by the opposite polarity microtubule motors kinesin and dynein in the embryos, and bind specifically to microtubules in-vitro. In the presence of ATP they exhibit long-range and short-range motility. For this proof-of-principle experiment I used fluorescently labeled AMPPNP, a non-hydrolysable analogue of ATP which binds to the motor domain of kinesin when microtubule-bound, to image the binding of the nucleotide to the motor and demonstrate the activity of the motors. While a large fraction of microtubule-bound droplets co-localized with a fluorescent AMPPNP molecule, non-specific binding of the nucleotide to the microscope slide surface prevented confirming the specificity of the colocalization events. Nevertheless, these data demonstrate the ability of the methodology to capture, in real time, the process of a regulator binding the motor at the single molecule level. / text

This thesis describes the host-guest chemistry between Cucurbit[7]uril (CB[7]) and CB[8] and a series of guests including bispyridinium cations, phenols and  napthalenes. These guests are bound to ruthenium polypyridine complexes or ruthenium based water oxidation catalysts (WOCs). The investigations are based upon utilizing the covalently linked photosensitizer and the electronic effects and chemical processes are investigated. / QC 20100927

abstract: The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

Orientador: Mário Tyago Murakami / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T22:14:14Z (GMT). No. of bitstreams: 1 Pinto_AlineSampaio_M.pdf: 3074349 bytes, checksum: 0fdb140ae2fd1c1975ba6cde0cf00bca (MD5) Previous issue date: 2012 / Resumo: As miosinas formam uma superfamília de proteínas de alto peso molecular com atividade mecanoquímica capaz de hidrolisar a molécula de ATP e de interagir com os filamentos de actina. A estrutura das miosinas pode ser divida de modo geral em cabeça motora, pescoço e cauda. São conhecidas 35 classes de miosinas em eucariotos sendo a classe II de miosinas denominada miosinas convencionais e as demais chamadas de não convencionais. Em plantas são somente encontradas as miosinas não convencionais de classe VIII e XI. A classe VIII caracteriza-se por sua alta processividade sobre os filamentos de actina e a classe XI é a maior classe em número de genes, tendo uma estrutura muito semelhante às miosinas de classe V. Uma terceira classe, a classe XIII, foi posteriormente descoberta e somente foi encontrada no gênero Acetabularia apresentando dois genes, porém essa classificação é controversa havendo aqueles que dizem que as miosinas da classe XIII possuem tanta afinidade filogenética com as da classe XI que elas deveriam compor uma única classe. As miosinas VIII e XI desempenham papéis chave no transporte direcional de componentes intracelulares em plantas, principalmente devido às grandes dimensões das células de plantas que não sobrevivem utilizando somente a difusão como mecanismo de transporte intracelular. Neste trabalho, buscamos selecionar os melhores representantes de cada classe a partir de análises in silico para desenvolvermos os testes de expressão, purificação e análises biofísicas. Os domínios selecionados, cauda globular (GT), dilute e SH3, foram clonados em pET28a e pET28aSUMO, os testes de expressão com diversas cepas de E coli, mostraram que o domínio dilute expressa em grande quantidade na fração solúvel, porém forma agregados impedindo as análises biofísicas e os ensaios de cristalização. O domínio SH3 também foi obtido na forma solúvel, porém em pouca quantidade e apresentou migração anômala no gel, sendo identificado a partir de análises de espectrometria de massas. Foram realizados testes iniciais de cristalização para o domínio SH3, mas não resultou na formação de cristais adequados a difração de raios X. A cauda globular foi obtida apenas na fração insolúvel e submetida a procedimentos de refolding para sua solubilização. Mesmo conseguindo o reenovelamento da construção, a mesma se manteve agregada inviabilizando os testes de cristalização. Por outro lado foram analisadas as interações da cauda globular da miosina XIh com presas identificadas por duplo-híbrido realizado pelo nosso grupo com a miosina humana Va. Três das proteínas que interagiram com a miosina Va humana também mostraram sinais de interação com a miosina XIh de Arabidopsis, indicando que mesmo havendo diferenças nas sequências polipeptídicas entre as classes de miosina, a estrutura terciária se mantém permitindo que ambas apresentem interações com algumas proteínas em comum / Abstract: Myosins belong to a superfamily of high molecular weight proteins, presenting mechanochemical activity by hydrolyzing ATP molecule and ability to interact with actin filaments. The myosin structure can be divided into motor head, neck and tail. 35 myosin classes are known in eukaryotic cells, where class II is known as conventional myosins and all others, as unconventional myosins. Plants possess only unconventional myosins including classes VIII and XI. The class VIII, is characterized by its high processivity on actin filaments while class XI possesses multiple genes and its protein structure is very similar to class V myosins. A third class, XIII, was later discovered and only found in Acetabularia genome presenting two genes. However this classification is controversial, because some studies shown that class XIII myosins share such high phylogenetic similarity with class XI that they should form the same class. Myosins VIII and XI play key roles on directional intracellular components transport in plants, mainly due to the large plant cell size which would not survive only by the diffusion mechanism for intracellular transport. In this work, we have selected the best representative targets of each class from in silico analyses to develop the protein expression, purification and biophysical characterization. The selected domains (globular tail (GT), dilute and SH3) were cloned into pET28a and pET28aSUMO expression vectors. Results of expression tests with several E coli strains, showed that dilute domain is expressed in large amounts in the soluble fraction; however, it aggregates preventing biophysical analysis and crystallization trials. The SH3 domain, expressed in low soluble concentration, presented an abnormal migration in SDS-PAGE and its identity was confirmed by mass spectrometry analysis. Initial crystallization tests were conducted with SH3 domain, but owing to the low protein concentration only clear drops have appeared, and no crystals or aggregates were observed. The globular tail domain, obtained only in insoluble fraction, was subjected to refolding procedures/ techniques in order to recover its native-like state; however, even the refolded protein displayed secondary structure, the protein remained aggregated. Furthermore, we analyzed the interactions between of myosin XIh globular tail with pre-identified proteins by yeast two-hybrid conducted by our group with human myosin Va. Three proteins that interacted with human myosin Va also showed interaction with Arabidopsis myosin XIh, indicating that despite of differences in polypeptide sequences between the classes XI and V, the tertiary structure may be maintained allowing both of them to have interactions with common proteins / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular