RESUMEN
Synthetic molecular machines are promising building blocks for future nanoscopic devices. However, the details of their mechanical behaviour are in many cases still largely unknown. A deeper understanding of mechanics at the molecular level is essential for the design and construction of complex nanodevices. Here, we show that transient two-dimensional infrared (T2DIR) spectroscopy makes it possible to monitor the conformational changes of a translational molecular machine during its operation. Translation of a macrocyclic ring from one station to another on a molecular thread is initiated by a UV pulse. The arrival of the shuttling macrocycle at the final station is visible from a newly appearing cross peak between these two moieties. To eliminate spectral congestion in the T2DIR spectra, we use a subtraction method applicable to many other complex molecular systems. The T2DIR spectra indicate that the macrocycle adopts a boat-like conformation at the final station, which contrasts with the chair-like conformation at the initial station.
RESUMEN
Small proteins provide good model systems for studying the fundamental forces that control protein folding. Here, we investigate the folding dynamics of the 28-residue zinc-finger mutant FSD-1, which is designed to form a metal-independent folded ßßα-motif, and which provides a testing ground for proteins containing a mixed α/ß fold. Although the folding of FSD-1 has been actively studied, the folding mechanism remains largely unclear. In particular, it is unclear in what stage of folding the α-helix is formed. To address this issue we investigate the folding mechanism of FSD-1 using a combination of temperature-dependent UV circular dichroism (UV-CD), Fourier transform infrared (FTIR) spectroscopy, two-dimensional infrared (2D-IR) spectroscopy, and temperature-jump (T-jump) transient-IR spectroscopy. Our UV-CD and FTIR data show different thermal melting transitions, indicating multistate folding behavior. Temperature-dependent 2D-IR spectra indicate that the α-helix is the most stable structural element of FSD-1. To investigate the folding/unfolding re-equilibration dynamics of FSD-1, the conformational changes induced by a nanosecond T-jump are probed with transient-IR and transient dispersed-pump-probe (DPP) IR spectroscopy. We observe biexponential T-jump relaxation kinetics (with time constants of 80 ± 13 ns and 1300 ± 100 ns at 322 K), confirming that the folding involves an intermediate state. The IR and dispersed-pump-probe IR spectra associated with the two kinetic components suggest that the folding of FSD-1 involves early formation of the α-helix, followed by the formation of the ß-hairpin and hydrophobic contacts.
Asunto(s)
Proteínas de Unión al ADN/química , Factores de Transcripción/química , Dicroismo Circular , Proteínas de Unión al ADN/genética , Cinética , Mutación , Pliegue de Proteína , Espectrofotometría Infrarroja , Temperatura , Factores de Tiempo , Factores de Transcripción/genética , Dedos de ZincRESUMEN
Here we follow, both experimentally and theoretically, the development of magnetism in Tb clusters from the atomic limit, adding one atom at a time. The exchange interaction is, surprisingly, observed to drastically increase compared to that of bulk, and to exhibit irregular oscillations as a function of the interatomic distance. From electronic structure theory we find that the theoretical magnetic moments oscillate with cluster size in exact agreement with experimental data. Unlike the bulk, the oscillation is not caused by the RKKY mechanism. Instead, the inter-atomic exchange is shown to be driven by a competition between wave-function overlap of the 5d shell and the on-site exchange interaction, which leads to a competition between ferromagnetic double-exchange and antiferromagnetic super-exchange. This understanding opens up new ways to tune the magnetic properties of rare-earth based magnets with nano-sized building blocks.
RESUMEN
AIMS: Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS: Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION: THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.
Asunto(s)
Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Microvasos/metabolismo , Neovascularización Patológica/metabolismo , Trombospondinas/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Placa Aterosclerótica/patología , Trombospondina 1/metabolismoRESUMEN
Rare-earth metals in their bulk form possess rather similar crystallographic structures, which is due to the very similar features of their outer electronic states. On the other hand, their magnetic properties are of rich variety, which is related to the specific form of the indirect magnetic exchange interaction between the inner electronic shells. In cluster form, this interplay may lead to very unusual magnetic structures. Here we show how the magnetic moments vary with size and temperature in Tm and Pr clusters. While in Pr clusters clear atom-by-atom oscillations indicate antiferromagnetic ordering, smooth variation and anomalous temperature behavior in Tm is representative for an essentially non-collinear spin arrangement. Their electric behavior is also very different, with a metallic-like behavior of Pr and localized electronic states in Tm.
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We present a simple method to measure the dynamics of cross peaks in time-resolved two-dimensional vibrational spectroscopy. By combining suitably weighted dispersed pump-probe spectra, we eliminate the diagonal contribution to the 2D-IR response, so that the dispersed pump-probe signal contains the projection of only the cross peaks onto one of the axes of the 2D-IR spectrum. We apply the method to investigate the folding dynamics of an alpha-helical peptide in a temperature-jump experiment and find characteristic folding and unfolding time constants of 260 ± 30 and 580 ± 70 ns at 298 K.
RESUMEN
Since the mid-nineties new waste incineration plants have come into operation in the Netherlands. Burning of waste can result in the emission of potentially toxic compounds. Although the incineration plants must comply with strict conditions concerning emission control, public concern on the possible impact on human health and the environment still exists. Multiple year (2004-2013) biomonitoring programs were set up around three waste incinerators for early detection of possible effects of stack emissions on the quality of crops and agricultural products. The results showed that the emissions did not affect the quality of crops and cow milk. Concentrations of heavy metals, PAHs and dioxins/PCBs were generally similar to background levels and did not exceed standards for maximum allowable concentrations in foodstuffs (e.g. vegetables and cow milk). Some exceedances of the fluoride standard for cattle feed were found almost every year in the maximum deposition areas of two incinerators. Biomonitoring with leafy vegetables can be used to monitor the real impact of these emissions on agricultural crops and to communicate with all stakeholders.
Asunto(s)
Productos Agrícolas/química , Dioxinas/análisis , Contaminantes Ambientales/análisis , Metales Pesados/análisis , Bifenilos Policlorados/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Alimentación Animal/análisis , Animales , Bovinos , Monitoreo del Ambiente/métodos , Contaminación de Alimentos/análisis , Incineración , Leche/química , Países Bajos , Poaceae/químicaRESUMEN
Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu- and Arg+ are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.