Effect of Osmolytes on Pressure‐Induced Unfolding of Proteins: A High‐Pressure SAXS Study

Herein, we explore the effect of different types of osmolytes on the high‐pressure stability and tertiary structure of a well‐characterized monomeric protein, staphylococcal nuclease (SNase). Changes in the denaturation pressure and the radius of gyration are obtained in the presence of different concentrations of trimethylamine N‐oxide (TMAO), glycerol and urea. To reveal structural changes in the protein upon compression at various osmolyte conditions, small‐angle X‐ray scattering (SAXS) experiments were carried out. To this end, a new high‐pressure cell suitable for high‐precision SAXS studies at synchrotron sources was built, which allows one to carry out scattering experiments up to maximum pressures of about 7 kbar. Our data clearly indicate that the osmolytes that stabilize proteins against temperature‐induced unfolding drastically increase their pressure stability and that the elliptically shaped curve of the pressure–temperature–stability diagram of proteins is shifted to higher temperatures and pressures with increasing osmolyte concentration. A drastic stabilization is observed for the osmolyte TMAO, which exhibits not only a significant stabilization against temperature‐induced unfolding, but also a particularly strong stabilization of the protein against pressure. In fact, such findings are in accordance with in vivo studies (for example P. J. Yancey, J. Exp. Biol. 2005 , 208, 2819–2830), where unusually high TMAO concentrations in some deep‐sea animals were found. Conversely, chaotropic agents such as urea have a strong destabilizing effect on both the temperature and pressure stability of the protein. Our data also indicate that sufficiently high TMAO concentrations might be able to largely offset the destabilizing effect of urea. The different scenarios observed are discussed in the context of recent experimental and theoretical studies.     

Osmolyte Effects on the Conformational Dynamics of a DNA Hairpin at Ambient and Extreme Environmental Conditions

The structural dynamics of a DNA hairpin (Hp) are studied in the absence and presence of the two natural osmolytes trimethylamine‐N‐oxide (TMAO) and urea at ambient and extreme environmental conditions, including high pressures and high temperatures, by using single‐molecule Förster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNA Hp is investigated on a single‐molecule level, providing novel mechanistic insights into its conformational conversions. Different from canonical DNA duplex structures of similar melting points, the DNA Hp is found to be rather pressure sensitive. The combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic, entropic, and volumetric contributions. The folded conformation is effectively stabilized by the compatible osmolyte TMAO not only at high temperatures, but also at high pressures and in the presence of the destabilizing co‐solute urea.     

Effect of Molecular Crowding on the Temperature–Pressure Stability Diagram of Ribonuclease A

FT‐IR spectroscopic and thermodynamic measurements were designed to explore the effect of a macromolecular crowder, dextran, on the temperature and pressure‐dependent phase diagram of the protein Ribonuclease A (RNase A), and we compare the experimental data with approximate theoretical predictions based on configuration entropy. Exploring the crowding effect on the pressure‐induced unfolding of proteins provides insight in protein stability and folding under cell‐like dense conditions, since pressure is a fundamental thermodynamic variable linked to molecular volume. Moreover, these studies are of relevance for understanding protein stability in deep‐sea organisms, which have to cope with pressures in the kbar range. We found that not only temperature‐induced equilibrium unfolding of RNase A, but also unfolding induced by pressure is markedly prohibited in the crowded dextran solutions, suggesting that crowded environments such as those found intracellularly, will also oppress high‐pressure protein unfolding. The FT‐IR spectroscopic measurements revealed a marked increase in unfolding pressure of 2 kbar in the presence of 30 wt % dextran. Whereas the structural changes upon thermal unfolding of the protein are not significantly influenced in the presence of the crowding agent, through stabilization by dextran the pressure‐unfolded state of the protein retains more ordered secondary structure elements, which seems to be a manifestation of the entropic destabilization of the unfolded state by crowding.     

On the Temperature–Pressure Free‐Energy Landscape of Proteins

We studied the thermodynamic stability of a small monomeric protein, staphylococcal nuclease (Snase), as a function of both temperature and pressure, and expressed it as a 3D free‐energy surface on the p,T‐plane using a second‐order Taylor expansion of the Gibbs free‐energy change ΔG upon unfolding. We took advantage of a series of different techniques (small‐angle Xray scattering, Fourier‐transform infrared spectroscopy, differential thermal analysis, pressure perturbation calorimetry and densitometry) in the evaluation of the conformation of the protein and in evaluating the changes in the thermodynamic parameters upon unfolding, such as the heat capacity, enthalpy, entropy, volume, isothermal compressibility and expansivity. The calculated results of the free‐energy landscape of the protein are in good agreement with experimental data of the p,T‐stability diagram of the protein over a temperature range from 200 to 400 K and at pressures from ambient pressure to 4000 bar. The results demonstrate that combined temperature–pressure‐dependent studies can help delineate the free‐energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. The approach presented may also be used for studying other systems with so‐called re‐entrant or Tamman loop‐shaped phase diagrams.     

Top-down tandem mass spectrometry of tRNA via ion trap collision-induced dissociation

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74–95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA^phe; rather, the mass was consistent with tRNA^Phe bearing an incomplete 3′-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5′- and 3′-termini of the molecule could be used to identify the major component as tRNA^phe-3′adenosine (without 3′-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA^Phe variant and the intact tRNA^Phe was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA^Phe-3′adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.     

Hydrolysis of tRNAPhe on Suspensions of Amino Acids

RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNA^Phe is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and β‐glutamic acid but not by suspensions of α‐glutamic acid, asparagine, or glutamine.     

The Synthesis of Methylated,Phosphorylated, and Phosphonated 3′‐Aminoacyl‐tRNASec Mimics

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA^Sec) in all domains of life. The multistep pathway involves O‐phosphoseryl‐tRNA:selenocysteinyl‐tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNA^Sec intermediate is converted into selenocysteinyl‐tRNA^Sec. The SepSecS architecture and the mode of tRNA^Sec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA^Sec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl‐derived tRNA^Sec mimics that contain a hydrolysis‐resistant ribose 3′‐amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site‐specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)‐2‐amino‐4‐phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three‐strand enzymatic ligation protocol to obtain the corresponding full‐length tRNA^Sec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS‐tRNA interactions. The novel tRNA^Sec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X‐ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA‐dependent enzymes.     

Enzymatic Synthesis of an RNA Fragment Corresponding to the Anti-Codon Loop and Stem of tRNAPhe from Yeast

Abstract

A hexadecamer corresponding to the anticodon loop and stem of tRNA^Phe yeast has been prepared using T₄ RNA ligase and isolated by high performance liquid chromatography. The two oligonucleotides used in the ligation were isolated from a ribonuclease T₁ digest of the tRNA which was resolved by HPLC on an anion exchange column. To prepare the “acceptor” oligonucleotide for the RNA ligase reaction a 3′ terminal phosphate was removed. To prepare the “donor” oligomer a 5′ terminal phosphate was added. Analysis of the product hexadecamer was by nucleoside and nucleotide-3′-mono-phosphate composition.     

Monitoring phase transition of aqueous biomass model substrates by high‐pressure and high‐temperature microfluidics

Aqueous‐Phase Reforming (APR) is a promising hydrogen production method, where biomass is catalytically reformed under high pressure and high temperature reaction conditions. To eventually study APR, in this paper, we report a high‐pressure and high‐temperature microfluidic platform that can withstand temperatures up to 200°C and pressures up to 30 bar. As a first step, we studied the phase transition of four typical APR biomass model solutions, consisting of 10 wt% of ethylene glycol, glycerol, xylose or xylitol in MilliQ water. After calibration of the set‐up using pure MilliQ water, a small increase in boiling point was observed for the ethylene glycol, xylitol and xylose solutions compared to pure water. Phase transition occurred through either explosive or nucleate boiling mechanisms, which was monitored in real‐time in our microfluidic device. In case of nucleate boiling, the nucleation site could be controlled by exploiting the pressure drop along the microfluidic channel. Depending on the void fraction, various multiphase flow patterns were observed simultaneously. Altogether, this study will not only help to distinguish between bubbles resulting from a phase transition and/or APR product formation, but is also important from a heat and mass transport perspective.     

Study of formation and thermal stability of the Fe/ZnO(000‐1) interface

Formation and thermal stability of the Fe/ZnO(000‐1) interface have been studied by means of X‐ray photoelectron spectroscopy and low energy electron diffraction. The results indicated a pseudo 2D growth mode for iron on ZnO. In addition, it could be shown that under ultra high vacuum conditions deposited Fe⁰ on a ZnO(000‐1) single crystal was partially oxidized by a small fraction of residual ? OH‐groups and ZnO to FeO. A strong temperature dependence of the interface reactivity was found upon annealing at temperatures up to 600 °C. Starting from 200 °C iron was first oxidized to bivalent iron oxide. After complete oxidation of Fe⁰ to Fe²⁺ at 375 °C, Fe²⁺ reacted to Fe³⁺. Above temperatures of 500 °C the deposited metallic iron was completely oxidized to trivalent iron. Further experiments with FeO on ZnO showed the oxidation state and the oxide film thickness of the deposited iron to be mainly dependent on the annealing temperature. Copyright © 2008 John Wiley & Sons, Ltd.     

Application of reference‐modified density functional theory: Temperature and pressure dependences of solvation free energy

Recently, we proposed a reference‐modified density functional theory (RMDFT) to calculate solvation free energy (SFE), in which a hard‐sphere fluid was introduced as the reference system instead of an ideal molecular gas. Through the RMDFT, using an optimal diameter for the hard‐sphere reference system, the values of the SFE calculated at room temperature and normal pressure were in good agreement with those for more than 500 small organic molecules in water as determined by experiments. In this study, we present an application of the RMDFT for calculating the temperature and pressure dependences of the SFE for solute molecules in water. We demonstrate that the RMDFT has high predictive ability for the temperature and pressure dependences of the SFE for small solute molecules in water when the optimal reference hard‐sphere diameter determined for each thermodynamic condition is used. We also apply the RMDFT to investigate the temperature and pressure dependences of the thermodynamic stability of an artificial small protein, chignolin, and discuss the mechanism of high‐temperature and high‐pressure unfolding of the protein. © 2017 Wiley Periodicals, Inc.     

Hydrostatic Pressure Increases the Catalytic Activity of Amyloid Fibril Enzymes

We studied the combined effects of pressure (0.1–200 MPa) and temperature (22, 30, and 38 °C) on the catalytic activity of designed amyloid fibrils using a high‐pressure stopped‐flow system with rapid UV/Vis absorption detection. Complementary FT‐IR spectroscopic data revealed a remarkably high pressure and temperature stability of the fibrillar systems. High pressure enhances the esterase activity as a consequence of a negative activation volume at all temperatures (about ?14 cm³ mol^?1). The enhancement is sustained in the whole temperature range covered, which allows a further acceleration of the enzymatic activity at high temperatures (activation energy 45–60 kJ mol^?1). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils.     

The Conformational Changes of 5SrRNA from Lupin Seeds and tRNAPhe in Presence of Ca2+, Mn2+ Cations by DSC Method

The results of calorimetric studies of 5SrRNA isolated from Lupinus luteus and of tRNA^Phe both in the absence and in the presence of different concentrations of cations Ca²⁺, Mn²⁺ were reported. The temperature and the enthalpy of melting were determined. Using the deconvolution method the elementary transitions were distinguished and discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

OH frequency shifts of fluorinated alcohols in nonpolar solvents for high pressures and low temperatures

Infrared spectra of fluorinated alcohols in nonpolar solvents were discussed in dependence on high pressures and low temperatures. The frequency of the monomeric OH stretching vibration, measured for the temperature at 20°C and for the pressure at 100 kPa, is about 20 cm⁻¹ lower than in the gas phase. The frequency decreases with increasing pressure up to 2 GPa. The frequency shift turns round for a further pressure increase. For higher than 8 GPa pressures the frequency is blue shifted in comparison to the gaseous state. The OH frequency decreases for a temperature change from 400 K up to 100 K. The frequency shift turns round for lower temperatures. This finding will be explained by a superposition of the unperturbed OH potential with Lennard-Jones function for different distances.     

A Covalent Approach for Site‐Specific RNA Labeling in Mammalian Cells

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site‐ and sequence‐specific covalent labeling of RNAs in mammalian cells by using tRNA^Ile2‐agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide‐bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site‐specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.     

Melting and dissociation of ammonia at high pressure and high temperature

Raman spectroscopy and synchrotron x-ray diffraction measurements of ammonia (NH(3)) in laser-heated diamond anvil cells, at pressures up to 60 GPa and temperatures up to 2500 K, reveal that the melting line exhibits a maximum near 37 GPa and intermolecular proton fluctuations substantially increase in the fluid with pressure. We find that NH(3) is chemically unstable at high pressures, partially dissociating into N(2) and H(2). Ab initio calculations performed in this work show that this process is thermodynamically driven. The chemical reactivity dramatically increases at high temperature (in the fluid phase at T > 1700 K) almost independent of pressure. Quenched from these high temperature conditions, NH(3) exhibits structural differences from known solid phases. We argue that chemical reactivity of NH(3) competes with the theoretically predicted dynamic dissociation and ionization.     

A Luminescent Metal–Organic Framework Thermometer with Intrinsic Dual Emission from Organic Lumophores

A new mixed‐ligand metal–organic framework (MOF), ZnATZ‐BTB, has been constructed as a luminescent ratiometric thermometer by making use of the intrinsic dual emission at cryogenic temperatures. Its twofold interpenetrated network promotes the Dexter energy transfer (DET) between the mixed organic lumophores. The temperature‐dependent luminescent behavior arises from the thermal equilibrium between two separated excited states coupled by DET, which is confirmed by Boltzmann distribution fitting. The small excited‐state energy gap allows ZnATZ‐BTB to measure and visualize cryogenic temperatures (30–130 K) with significantly high relative sensitivity (up to 5.29 % K^?1 at 30 K). Moreover, it is the first example of a ratiometric MOF thermometer the dual emitting sources of which are widely applicable mixed organic ligands, opening up new opportunities for designing such devices.