Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function

Ion and Water Retention by Permeabilized Cells

Nonionic detergents, Triton X-100 and Brij 58, removed, within 2-5 minutes, lipid membrane of suspended thymus lymphocytes and monolayer H-50 cells grown in culture. Studies of hydration, ionic asymmetry, and ionic and protein release kinetics were conducted on these membraneless cellular preparations. The hydration of nuclei isolated by Triton X-100 procedures appears to be influenced strongly by the monovalent ionic concentration of the buffer bathing the organelles. The putative monovalent ionic concentration of the cellular aqueous phase (i.e., 150 meq/L) caused nuclei to swell and coalesce. Monovalent ionic concentrations of 30 meq/L or less caused minimal changes in volume and in morphology. Triton X-100 treatment led to rapid mobilization and solubilization of membrane and cytoplasmic lipids and proteins, and the cellular potassium was reduced to very low levels. Brij 58 treatment of the lymphocytes for 5 minutes led to loss of membrane structure. Potassium, however, was retained at significant levels for over 10 minutes. Potassium and protein release kinetic studies in the H-50 monolayer cells following Brij treatment revealed that potassium and the detergent mobilized proteins may be co-compartmentalized and that 10 minutes or more are required before their release is completed. These results support the view that most of the potassium and diffusible proteins are not fully dissolved in the cellular water. Furthermore, the integrity of the membranes does not appear to be essential for the retention of the ions and the proteins

The State of Potassium in Skeletal Muscle and in Non-Muscle Cells

The relationship between ions, water, and the electrical properties are fundamental to our understanding of cellular function. This paper is primarily directed at reviewing the theoretical explanations for the changes in cellular potential and ionic composition which are associated with early postnatal development of skeletal muscle. The findings are: (a) a two-fold reduction in tissue hydration and a significant reduction of the diffusive motion of cellular water; (b) ten-fold decrease in cellular sodium; (c) six-fold decrease in tissue chloride; (d) the concentrations of intracellular potassium, and of extracellular sodium, potassium and chloride were constant; and (e) the cellular potential changed by 55 mV. A review of the literature concerning the physical state of potassium and water is made. The theoretical explanations of these findings are evaluated in terms of the classical membrane theory and the association induction hypothesis

Transthyretin amiloid fibrillumok nanobiofizikai vizsgálata = Nanobiophysical exploration of transthyretin amyloid fibrils

Kísérleteinkben AFM segítségével vizsgáltuk transthyretin (TTR) amiloid fibrillumok amiloidogén fibrillum képződési mechanizmusait. Protofibrillumokon végzett egyedi molekula erőspektroszkópiai mérések eredményeit a natív TTR szerkezeti paramétereivel hasonlítottuk össze annak érdekében, hogy szerkezeti és dinamikai bepillantást nyerjünk a fibrillumok belső elrendezésébe és az összetartó erők természetébe. Időfüggő AFM felvételek segítségével a protofibrillum képződést követő belső szerkezeti változásokat térképeztük fel. Eredményeink szerint a protofibrillum képződés első lépése amorf aggregátumok kialakulása, amelyek idővel gyűrű alakú szerkezetekké állnak össze. Hasonló gyűrű alakú intermediéreket más amiloid fibrillumok esetében is megfigyeltek. A gyűrűk egymáshoz rendeződve tubuláris strukturákat alakítanak ki, amely a protofibrillum kialakulásának további alapját képezi. Az oldat kicserélésére a struktura szétesik, szétzippzározódási lépéseken keresztül. A nanomechanikai mérések arra utalnak, hogy a TTR egységek ß-szálak mentén tekerednek szét, továbbá érett protofibrillumokban az intermonomerikus kapcsolatok megerősödnek. Megfigyeléseink alapján a TTR fibrillogenezis egy szerkezeti modelljét állítottuk fel. | In this work we used AFM to follow the amyloidogenetic pathway of transthyretin (TTR) by imaging the events leading to the formation of amyloid protofilaments. Single-molecule force spectroscopy (SMFS) of protofilaments was compared to naive TTR in order to probe dynamic and structural differences. We observed that the pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers (rings or doughnuts). In other types of amyloidoses similar ring structures have been implicated in cytoxicity, but their properties and involvement in the amyloid pathway are poorly understood. We show that the rings have a tendency to stack, forming tubular protofilaments. These tubular protofilaments precede the appearance of amyloid protofilaments. Their height and pitch resemble those of previous structural models for the TTR amyloid protofilament. Upon solvent exchange we also observed amyloid protofilament dissociation. The dissociation appears to proceed through an unzipping mechanism, revealing structures reminiscent of the TTR annular oligomers. SMFS of protofilaments revealed a time-dependent increase in the length of the manipulated structure, suggesting that associations between monomers stabilize with time. Force spectra of native TTR and protofilaments contained transitions spaced 4 nm apart, indicating that the component ß-strands unfold sequentially. Based on our results a model of TTR protofilament assembly is proposed

Release of Hemoglobin and Potassium from Human Red Blood Cells Treated with Triton X-100 Under the Critical Micellar Concentration

The action of detergents is thought to be connected primarily with micelle formation. However, detergent monomers can also effect biological systems. It was found in this study that human red blood cells can be disintegrated with Triton X-100 non-ionic detergent at a concentration of 0.007 %, lower than the critical micellar concentration (CMC). The time dependent release of hemoglobin and potassium was detected at 37°C and both were sigmoid in character. Although potassium was released faster than hemoglobin, a cooperative relationship between potassium and hemoglobin within the intact red blood cell is suggested by this observation

Intracelluláris kalcium transzporterek vizsgálata élesztő (Saccharomyces cerevisiae) modellben

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Stretching Single Domain Proteins: Phase Diagram and Kinetics of Force-Induced Unfolding

Single molecule force spectroscopy reveals unfolding of domains in titin upon stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a step-wise "quantized" manner. Unfolding dynamics depends sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value which is determined by the barrier to unfolding when f=0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.Comment: 12 pages, Latex, 6 ps figure

Dynamic Strength of Titin's Z-Disk End

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment

Selective lactase deficiency is common in pediatric patients undergoing upper endoscopy

Lactase deficiency can lead to significant symptoms in the pediatric population. To date, few studies have examined the prevalence of enzyme testing-based lactase and other disaccharidase deficiencies (DDs) in pediatric patients undergoing upper endoscopic evaluation. The primary objective of this study was to determine the prevalence of selective lactase and other DDs amongst a large cohort of pediatric patients with and without inflammatory bowel disease (IBD: Crohn’s disease and ulcerative colitis) via a chart review of 739 patients who underwent esophago-gastro-dudenoscopy EGD between April 2010 and August 2016. We identified 560 pediatric patients (ages 1-18 years) who underwent mucosal enzyme testing at the time of their EGD. The overall rate of lactase deficiency (LD) was 39%. LD positively correlated with age (p=0.00017), but there was no significant difference between age matched IBD and non-IBD patients (45% vs. 42% p=0.68). Four patients (0.17%) were found to have selective maltase deficiency. No selective sucrase or palatinase deficiency was identified. Statistically significant differences occurred in lactase deficiency amongst patients of different races. In conclusion, lactase deficiency is a relatively common finding in children undergoing EGD though at no increased rate amongst the IBD patient population. Disaccharidase testing should be considered in pediatric patients undergoing EGD