Protein folding on the ribosome studied using NMR spectroscopy

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome-nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity

Systematic mapping of free energy landscapes of a growing filamin domain during biosynthesis

Cotranslational folding (CTF) is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of CTF pathways. Here, we correlate observations of translationally arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome, and thus, quantify a substantial destabilization of the native structure on the ribosome. We identify and characterize two folding intermediates formed in isolation, including a partially folded intermediate associated with the isomerization of a conserved cis proline residue. The slow folding associated with this process raises the prospect that neighboring unfolded domains might accumulate and misfold during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation and show that catalysis of folding by peptidyl-prolyl isomerases is sufficient to eliminate this hazard. [Abstract copyright: Copyright © 2018 the Author(s). Published by PNAS.

The effects of hydrology on plankton biomass in shallow lakes of the Pampa Plain

Climatic and hydrological variability is usually high in the Pampa Plain (Argentina). However it has not studied yet how this variability may affect the phytoplankton and zooplankton biomass and community structure in aquatic systems of this region. The main purpose of this study was to assess flushing effects on nutrient and plankton dynamics in two interconnected very shallow lakes of the Pampa Plain. In order to study the impact of hydrology on the plankton biomass and community structure, we compared the summer plankton community among three consecutive years with contrasting hydrological characteristics. Water residence time varied an order of magnitude among years and this variability was correlated to strong changes in physicochemical and biological lake characteristics. Depending on the water discharge level, the hydrological regime within the lakes ranged from lentic to more lotic conditions. Nutrient and phytoplankton biomass were positively related to water discharges. During high flushing periods, nutrients import from intensive agriculture lands leads to a dramatic increase in trophic conditions. On the other hand, macrozooplankton biomass was positively related to water residence time and showed a dramatic decrease during high flushing years. Rotifers biomass was not affected by interannual water discharge variability during the study period. Our results support that in case of lakes with high flushing rates, zooplankton development is dependent on water residence time and that hydrology may have stronger effects on macrozooplankton biomass than top-down control by planktivores.Fil: Rennella, A. M.. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Sistemas de Producción Acuatica; ArgentinaFil: Quiros, Rolando. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Sistemas de Producción Acuatica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentin

A Numerical Study on a Ceramic Matrix composite Wing Leading Edge

The design of the wing leading edge of re-entry vehicles is a very challenging task since severe aerothermal loads are encountered during the re-entry trajectory. Hence, advanced materials and structural concepts need to be adopted to withstand the elevated thermal gradients and stresses. Furthermore, particular attention must be paid to the design of hot areas and connections between hot and cold areas of the structure, where the presence of major thermal gradients associated to significant thermal expansion coefficients variations, can lead to damage onset and failure. In order to face this issues, Ceramic Matrix Composites are generally employed as passive hot structures due of their capability to operate at elevated temperatures retaining acceptable mechanical properties. In the present work a novel thermo-structural concept of an hypersonic wing leading edge is introduced and verified by means of and advanced finite element thermo-structural model

Determining the energy landscape of proteins by a fast isotope exchange NMR approach

ABSTRACT: We present a new and efficient NMR method, BLUU-Tramp (Biophysics Laboratory University of Udine temperature ramp), for the collection of hydrogen 12deuterium exchange experiments as a function of time and temperature for small and medium-sized proteins. Exchange rates can be determined to extract the underlying thermodynamic equilibrium or kinetic param- eters by sampling hundreds of points over a virtually continuous temperature ramp. Data are acquired in a single experimental session that lasts some 20 1260 h, depending on the thermal stability of the protein. Subsequent analysis provides a complete thermodynamic description of the protein energy landscape. The global thermal unfolding process and the partial or local structure opening events can be fully determined at the single- residue resolution level. The proposed approach is shown to work successfully with the amyloidogenic protein \u3b22- microglobulin. With 15N-labeling, the unfolding landscape of a protein can also be studied in the presence of other unlabeled proteins and, in general, with ligands or cosolutes or in physiological environments

Single-shot NMR measurement of protein unfolding landscapes.

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine-Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is (15)N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein β(2)-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics, Thermodynamics, and Structure

International audienceThe transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration