It has been shown that sodium salts significantly affect the temperature of the coil-to-globule collapse transition of poly(N-isopropylacrylamide) J. Am. Chem. Soc., 2005, 127, 14505. Since this ...phenomenon resembles the cold renaturation of globular proteins, it can be studied by means of the theoretical approach devised to rationalise the occurrence and the mechanism of cold denaturation G. Graziano, Phys. Chem. Chem. Phys., 2010, 12, 14245; Phys. Chem. Chem. Phys., 2014, 16, 21755. It emerges that the collapse transition is driven by the decrease in the solvent-excluded volume in order to maximise the translational entropy of water molecules and ions. At a given temperature, the aqueous solutions of sodium salts have densities higher than that of water. For this reason, the magnitude of the solvent-excluded volume effect proves to be larger, stabilizing the globular conformations of poly(N-isopropylacrylamide). On the other hand, two large ions, iodide and thiocyanate, are poorly hydrated and stabilise the coil conformations of the polymer by a preferential binding mechanism.
We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid‐state NMR spectroscopy with 100–111 kHz magic‐angle spinning (MAS). The excellent resolution in the Cα‐Hα ...plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα detection block was developed and applied for the sequence‐specific backbone and aliphatic side‐chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.
No deuterium: With new 111 kHz magic‐angle spinning probes, high‐resolution 1H‐detected NMR spectroscopy of insoluble, crystalline, or self‐assembled protein aggregates is now feasible without replacing side‐chain protons with deuterons. α‐Protons become sensitive and spectrally resolved NMR probes, which allow backbone and side‐chain resonance assignment in about one week of experimental time for proteins of about 20 kDa.
Elastin-like polypeptides (ELPs) are soluble in water at low temperature, but, on increasing the temperature, they undergo a reversible and cooperative, coil-to-globule collapse transition. It has ...been shown that the addition to water of either trimethylamine
-oxide (TMAO), glycine, or betaine causes a significant decrease of T(collapse) in the case of a specific ELP. Traditional rationalizations of these phenomena do not work in the present case. We show that an alternative approach, grounded in the magnitude of the solvent-excluded volume effect and its temperature dependence (strictly linked to the translational entropy of solvent and co-solute molecules), is able to rationalize the occurrence of ELP collapse in water on raising the temperature, as well as the T(collapse) lowering caused by the addition to water of either TMAO, glycine, or betaine.
Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In ...particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.
Both sodium chloride and sodium sulfate are able to stabilize yeast frataxin, causing an overall increase of its thermodynamic stability curve, with a decrease in the cold denaturation temperature ...and an increase in the hot denaturation one. The influence of low concentrations of these two salts on yeast frataxin stability can be assessed by the application of a theoretical model based on scaled particle theory. First developed to figure out the mechanism underlying cold denaturation in water, this model is able to predict the stabilization of globular proteins provided by these two salts. The densities of the salt solutions and their temperature dependence play a fundamental role.
Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and ...exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.
Thrombin plays a pivotal role in the coagulation cascade; therefore, it represents a primary target in the treatment of several blood diseases. The 15‐mer DNA oligonucleotide 5′‐GGTTGGTGTGGTTGG‐3′, ...known as thrombin binding aptamer (TBA), is a highly potent inhibitor of the enzyme. TBA folds as an antiparallel chair‐like G‐quadruplex structure, with two G‐tetrads surrounded by two TT loops on one side and a TGT loop on the opposite side. Previous crystallographic studies have shown that TBA binds thrombin exosite I by its TT loops, T3T4 and T12T13. In order to get a better understanding of the thrombin–TBA interaction, we have undertaken a crystallographic characterization of the complexes between thrombin and two TBA mutants, TBAΔT3 and TBAΔT12, which lack the nucleobase of T3 and T12, respectively. The structural details of the two complexes show that exosite I is actually split into two regions, which contribute differently to TBA recognition. These results provide the basis for a more rational design of new aptamers with improved therapeutic action.
The X‐ray structure of two mutants of thrombin binding aptamer (TBA) in complex with their target protein shed new light about the recognition process. The interaction site of thrombin (exosite I) is split into two regions differently contributing to the binding. This result suggests new TBA modifications to improve the aptamer action.
The SARS-CoV-2 infection is now a part of the everyday lives of immunocompromised patients, but the choice of treatment and the time of viral clearance can often be complex, exposing patients to ...possible complications. The role of the available antiviral and monoclonal therapies is a matter of debate, as are their effectiveness and potential related adverse effects. To date, in the literature, the amount of data on the use of combination therapies and on the multiple lines of anti-SARS-CoV-2 therapy available to the general population and especially to inborn error of immunity (IEI) patients is small.
Here, we report a case series of five adult IEI patients managed as inpatients at three Italian IEI referral centers (Rome, Treviso, and Cagliari) treated with combination therapy or multiple therapeutic lines for SARS-CoV-2 infection, such as monoclonal antibodies (mAbs), antivirals, convalescent plasma (CP), mAbs plus antiviral, and CP combined with antiviral.
This study may support the use of combination therapy against SARS-CoV-2 in complicated IEI patients with predominant antibody deficiency and impaired vaccine response.
Cardiac resynchronization therapy (CRT) is usually performed with biventricular pacing (BiVP), but recently, conduction system pacing (CSP) has been proposed as an alternative in case of BiVP ...failure. The aim of this study is to define an algorithm to choose between BiVP and CSP resynchronization using the interventricular conduction delays (IVCD) as a guide.
Consecutive patients from January 2018 to December 2020 with an indication for CRT were prospectively enrolled in the study group (delays-guided resynchronization group, DRG). A treatment algorithm based on IVCD was used to decide whether to leave the left ventricular (LV) lead to perform BiVP or pull it out and perform CSP. Outcomes from the DRG group were compared to a historical cohort of CRT patients who underwent CRT procedures between January 2016 and December 2017 (resynchronization standard guide group, SRG). The primary endpoint was a composite of cardiovascular mortality, heart failure (HF) hospitalization, or HF event at 1 year after the date of intervention.
The study population consisted of 292 patients, of which 160 (54.8%) were in the DRG and 132 (45.2%) in the SRG. In the DRG, 41 of 160 patients underwent CSP based on the treatment algorithm (25.6%). The primary endpoint was significantly higher in the SRG (48/132, 36.4%) compared to the DRG (35/160, 21.8%) (hazard ratio (HR): 1.72; 95% confidence interval (CI): 1.12-2.65;
= 0.013).
A treatment algorithm based on IVCD shifted one patient out of every four from BiVP to CSP, with consequent reduction in the primary endpoint after implantation. Therefore, its application could be useful to determine whether to perform BiVP or CSP.
An entropic stabilization mechanism has recently gained attention and credibility as the physical ground for the extra thermal stability of globular proteins from thermophilic microorganisms. An ...empirical result, obtained from the analysis of thermodynamic data for a large set of proteins, strengthens the general reliability of the theoretical approach originally devised to rationalize the occurrence of cold denaturation Graziano, PCCP 2014, 16, 21755–21767. It is shown that this theoretical approach can readily account for the entropic stabilization mechanism. On decreasing the conformational entropy gain associated with denaturation, the thermal stability of a model globular protein increases markedly.
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