The interaction of thiocyanate with peptides-A computational study.

According to the Hofmeister series, thiocyanate is the strongest "salting in" anion. In fact, it has a strong denaturant activity against the native state of globular proteins. A molecular level rationalization of the Hofmeister series is still missing, and therefore the denaturant activity of thiocyanate also awaits a robust explanation. In the last years, different types of experimental studies have shown that thiocyanate is capable to directly interact with both polar and nonpolar groups of polypeptide chains. This finding has been scrutinized via a careful computational procedure based on density functional theory approaches. The results indicate that thiocyanate is able to make H-bonds via both the nitrogen and sulfur atom, and to make strong van der Waals interactions with almost all the groups of polypeptide chains, regardless of their polarity.

The Action of Chemical Denaturants: From Globular to Intrinsically Disordered Proteins.

Proteins perform their many functions by adopting either a minimal number of strictly similar conformations, the native state, or a vast ensemble of highly flexible conformations. In both cases, their structural features are highly influenced by the chemical environment. Even though a plethora of experimental studies have demonstrated the impact of chemical denaturants on protein structure, the molecular mechanism underlying their action is still debated. In the present review, after a brief recapitulation of the main experimental data on protein denaturants, we survey both classical and more recent interpretations of the molecular basis of their action. In particular, we highlight the differences and similarities of the impact that denaturants have on different structural classes of proteins, i.e., globular, intrinsically disordered (IDP), and amyloid-like assemblies. Particular attention has been given to the IDPs, as recent studies are unraveling their fundamental importance in many physiological processes. The role that computation techniques are expected to play in the near future is illustrated.

A Structure-Based Mechanism for the Denaturing Action of Urea, Guanidinium Ion and Thiocyanate Ion.

An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate.

Atomic-Level View of the Functional Transition in Vertebrate Hemoglobins: The Case of Antarctic Fish Hbs.

Tetrameric hemoglobins (Hbs) are prototypal systems for studies aimed at unveiling basic structure-function relationships as well as investigating the molecular/structural basis of adaptation of living organisms to extreme conditions. However, a chronological analysis of decade-long studies conducted on Hbs is illuminating on the difficulties associated with the attempts of gaining functional insights from static structures. Here, we applied molecular dynamics (MD) simulations to explore the functional transition from the T to the R state of the hemoglobin of the Antarctic fish Trematomus bernacchii (HbTb). Our study clearly demonstrates the ability of the MD technique to accurately describe the transition of HbTb from the T to R-like states, as shown by a number of global and local structural indicators. A comparative analysis of the structural states that HbTb assumes in the simulations with those detected in previous MD analyses conducted on HbA (human Hb) highlights interesting analogies (similarity of the transition pathway) and differences (distinct population of intermediate states). In particular, the ability of HbTb to significantly populate intermediate states along the functional pathway explains the observed propensity of this protein to assume these structures in the crystalline state. It also explains some functional data reported on the protein that indicate the occurrence of other functional states in addition to the canonical R and T ones. These findings are in line with the emerging idea that the classical two-state view underlying tetrameric Hb functionality is probably an oversimplification and that other structural states play important roles in these proteins. The ability of MD simulations to accurately describe the functional pathway in tetrameric Hbs suggests that this approach may be effectively applied to unravel the molecular and structural basis of Hbs exhibiting peculiar functional properties as a consequence of the environmental adaptation of the host organism.

Some Clues about Enzymes from Psychrophilic Microorganisms.

Enzymes purified from psychrophilic microorganisms prove to be efficient catalysts at low temperatures and possess a great potential for biotechnological applications. The low-temperature catalytic activity has to come from specific structural fluctuations involving the active site region, however, the relationship between protein conformational stability and enzymatic activity is subtle. We provide a survey of the thermodynamic stability of globular proteins and their rationalization grounded in a theoretical approach devised by one of us. Furthermore, we provide a link between marginal conformational stability and protein flexibility grounded in the harmonic approximation of the vibrational degrees of freedom, emphasizing the occurrence of long-wavelength and excited vibrations in all globular proteins. Finally, we offer a close view of three enzymes: chloride-dependent α-amylase, citrate synthase, and ß-galactosidase.

A Protein Data Bank survey of multimodal binding of thiocyanate to proteins: Evidence for thiocyanate promiscuity.

Over the last one and half century, a myriad of studies has demonstrated that Hofmeister ions have a major impact on protein stability and solubility. Nevertheless, the definition of the physico-chemical basis of their activity has proved to be highly challenging and controversial. Here, by exploiting the enormous information content of the Protein Data Bank, we explored the binding to proteins of thiocyanate, the anion of the series exerting the highest solubilization/destabilization effects. The survey, which led to the identification and characterization of 712 thiocyanate binding sites, provides a comprehensive and atomic-level view of the varied interactions that the ion forms with proteins. The inspection of these sites highlights a limited tendency of thiocyanate to interact with structured water molecules, in line with the reported poor hydration of the ion. On the other hand, the thiocyanate makes interactions with protein nonpolar moieties, especially with the backbone Cα atom. In as many as 104 cases, the ion exclusively makes nonpolar contacts. In conclusion, these findings suggest that the ability of thiocyanate to bind all types of protein exposed patches may lead to the formation of a negatively charged electrostatic barrier that could prevent protein-protein aggregation and promote protein solubility. Moreover, the denaturing action of thiocyanate may be ascribed to its ability to establish multiple attractive interactions with protein surfaces.

A Rationalization of the Effect That TMAO, Glycine, and Betaine Exert on the Collapse of Elastin-like Polypeptides.

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 N-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.

General Counteraction Exerted by Sugars against Denaturants.

The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native state, while others stabilize it. It is emerging that stabilizing agents are able to counteract the action of destabilizing agents. We have already provided experimental evidence that this counteraction is a general phenomenon and offered a rationalization. In the present work, we show that four different sugars, namely fructose, glucose, sucrose, and trehalose, counteract the effect of urea, tetramethylurea, sodium perchlorate, guanidinium chloride, and guanidinium thiocyanate despite the chemical and structural differences of those destabilizing agents. The rationalization we provide is as follows: (a) the solvent-excluded volume effect, a purely entropic effect, stabilizes the native state, whose solvent-accessible surface area is smaller than the one of denatured conformations; (b) the magnitude of the solvent-excluded volume effect increases markedly in ternary solutions because the experimental density of such solutions is larger than that of pure water.

Comment on "The Gibbs free energy of cavity formation in a diverse set of solvents"[J. Chem. Phys. 153, 134501 (2020)].

It is pointed out that the unexpected result that the magnitude of the reversible work of cavity creation in ethylene glycol proves to be larger than that in water [I. Sedov and T. Magsumov, J. Chem. Phys. 153, 134501 (2020)] could be due to that (a) the density of the used computational model of this liquid is "significantly" larger than the experimental one and (b) the procedure adopted to perform the comparison among the different liquids is not "strictly" correct. It is also indicated that several lines of evidence suggest that the magnitude of the reversible work of cavity creation in water can be larger than that in ethylene glycol.

Can the roles of polar and non-polar moieties be reversed in non-polar solvents?

Using thermodynamic integration, we study the solvation free energy of 18 amino acid side chain equivalents in solvents with different polarities, ranging from the most polar water to the most non-polar cyclohexane. The amino acid side chain equivalents are obtained from the 20 natural amino acids by replacing the backbone part with a hydrogen atom, and discarding proline and glycine that have special properties. A detailed analysis of the relative solvation free energies suggests how it is possible to achieve a robust and unambiguous hydrophobic scale for the amino acids. By discriminating the relative contributions of the entropic and enthalpic terms, we find strong negative correlations in water and ethanol, associated with the well-known entropy-enthalpy compensation, and a much reduced correlation in cyclohexane. This shows that in general the role of the polar and non-polar moieties cannot be reversed in a non-polar solvent. Our findings are compared with past experimental as well as numerical results, and may shed additional light on the unique role of water as a biological solvent.

Why small proteins tend to have high denaturation temperatures.

Data indicate that small globular proteins (consisting of less than about 70 residues) tend to have high denaturation temperatures. This finding is analysed by comparing experimental denaturation enthalpy and entropy changes of a selected set of small proteins with values calculated on the basis of average and common properties of globular proteins. The conclusion is that the denaturation entropy change is smaller than expected, leading to an increase in denaturation temperature. The proposed molecular rationalization considers the existence of long-wavelength, low-frequency vibrational modes in the native state of small proteins due to their large surface-to-interior ratio. The effect of decreasing the conformational entropy gain associated with denaturation on thermal stability is directly verified by means of an already devised theoretical model [G. Graziano, Phys. Chem. Chem. Phys. 2010, 12, 14245-14252; 2014, 16, 21755-21767].

Guanidinium binding to proteins: The intriguing effects on the D1 and D2 domains of Thermotoga maritima Arginine Binding Protein and a comprehensive analysis of the Protein Data Bank.

Thermotoga maritima Arginine Binding Protein has been extensively characterized because of its peculiar features and its possible use as a biosensor. In this characterization, deletion of the C-terminal helix to obtain the monomeric protein TmArgBP20-233 and dissection of the monomer in its two domains, D1 and D2, have been performed. In the present study the stability of these three forms against guanidinium chloride is investigated by means of circular dichroism and differential scanning calorimetry measurements. All three proteins show a high conformational stability; moreover, D1 shows an unusual behavior in the presence of low concentrations of guanidinium chloride. This finding has led us to investigate a possible binding interaction by means of isothermal titration calorimetry and X-ray crystallography; the results indicate that D1 is able to bind the guanidinium ion (GuH+), due to its similarity with the arginine terminal moiety. The analysis of the structural and dynamic properties of the D1-GuH+ complex indicates that the protein binds the ligand through multiple and diversified interactions. An exhaustive survey of the binding modes of GuH+ to proteins indicates that this is a rather common feature. These observations provide interesting insights into the effects that GuH+ is able to induce in protein structures.

On the extraordinary pressure stability of the Thermotoga maritima arginine binding protein and its folded fragments - a high-pressure FTIR spectroscopy study.

The arginine binding protein from T. maritima (ArgBP) exhibits several distinctive biophysical and structural properties. Here we show that ArgBP is also endowed with a ramarkable pressure stability as it undergoes minor structural changes only, even at 10 kbar. A similar stability is also observed for its folded fragments (truncated monomer and individual domains). A survey of literature data on the pressure stability of proteins highlights the uncommon behavior of ArgBP.

Hydrophobic hydration and pairwise hydrophobic interaction of Lennard-Jones and Mie particles in different water models.

The study provides a deep computational analysis of the thermodynamic and structural features associated with the hydration of xenon, Xe, and its pairwise hydrophobic interaction (i.e., the potential of mean force, PMF), over a large temperature range. Xe is described both as a Lennard-Jones particle, LJ-Xe, and as a Mie particle, Mie-Xe (pseudo hard sphere). Three different water models are used: TIP3P-Ew, SPCE and TIP4P-2005. Mie-Xe is more hydrophobic than LJ-Xe due to the lack of the attractive energetic interactions with water molecules; its hydration, around room temperature, is opposed by a large and negative entropy change and a positive enthalpy change. The PMF of Mie-Xe is characterized by a deep minimum at contact distance whose depth increases with temperature, and whose magnitude is significantly larger than that obtained for LJ-Xe. The contact minimum configuration of Mie-Xe is favoured by a large positive entropy change and contrasted by a positive enthalpy change. These results are qualitatively the same regardless of the water model used. There is no clear connection between the values determined for the thermodynamic functions and the structural features of the hydration shells surrounding the single Mie-Xe and the couple of Mie-Xe particles in the contact minimum configuration. This confirms that the structural reorganization of water associated with such processes is characterized by an almost complete enthalpy-entropy compensation.

Effect of sodium thiocyanate and sodium perchlorate on poly(N-isopropylacrylamide) collapse.

The T(collapse) of poly(N-isopropylacrylamide), PNIPAM, shows a nonlinear dependence on the concentration of NaSCN or NaClO4; in the case of NaClO4, for example, at very low concentrations of the salt, T(collapse) increases with the concentration, while it has an opposite trend at higher NaClO4 concentrations [J. Am. Chem. Soc., 2005, 127, 14505]. These puzzling experimental data can be rationalized by considering that low charge density and poorly hydrated ions, such as thiocyanate and perchlorate, interact preferentially with the surface of the polymer, and cause an increase of the magnitude of the energetic term that stabilizes swollen conformations at low salt concentrations. However, as both swollen and collapsed PNIPAM conformations are accessible to such ions in view of their large conformational freedom, the difference in the number of ions bound to PNIPAM surface upon collapse changes little on increasing the salt concentration. Thus, the energetic term that favors swollen conformations increases with salt concentration to a lesser extent than the solvent-excluded volume term (linked to the density increase caused by salt addition to water), that favors collapsed conformations, leading to a nonlinear trend of T(collapse).

Contrasting the hydration thermodynamics of methane and methanol.

Experimental hydration thermodynamic functions of methane and methanol over a large temperature range and at 1 atm (coming from previous studies of others) are analysed by means of a theoretical approach grounded in statistical mechanics. It is shown that the hydration Gibbs energy change can be reproduced quite well for both solutes by adding the large positive reversible work of cavity creation to the solute-water van der Waals and H-bond energetic attractions. The large negative hydration entropy change of both methane and methanol comes from the decrease in translational entropy of water molecules due to the solvent-excluded volume effect caused by cavity creation in water. The reorganization of water-water H-bonds upon solute insertion is characterized by almost complete enthalpy-entropy compensation and so cannot affect the hydration Gibbs energy change. In addition, such reorganization is endothermic above 4 °C, suggesting that no significant increase in water structure occurs upon methane or methanol insertion.

[The new National Guidelines System in Italy: a first evaluation].

The so-called Gelli-Bianco Law (L 24/2017) recognizes the central role of Guidelines as far as decision-making in the health sector is concerned and establishes the Italian National Guidelines System (SNLG) as a "safe harbor", notwithstanding the specificities of each real-world case. The Italian National Center for Clinical Excellence, Quality and Security (CNEC) is responsible for the creation and the development of the SNLG. After considering the many issues revolving around the creation of shared Guidelines, both on the national and international level, we present here the activities of the SNLG to date, together with an evaluation of how the new rules and practices around the creation of National Guidelines have been received. We also provide a series of recommendations for scientific societies on how best to produce Guidelines in their own field and how to submit them to the SNLG.

The characterization of Thermotoga maritima Arginine Binding Protein variants demonstrates that minimal local strains have an important impact on protein stability.

The Ramachandran plot is a versatile and valuable tool that provides fundamental information for protein structure determination, prediction, and validation. The structural/thermodynamic effects produced by forcing a residue to adopt a conformation predicted to be forbidden were here explored using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model. Specifically, we mutated TmArgBP Gly52 that assumes a conformation believed to be strictly disallowed for non-Gly residues. Surprisingly, the crystallographic characterization of Gly52Ala TmArgBP indicates that the structural context forces the residue to adopt a non-canonical conformation never observed in any of the high-medium resolution PDB structures. Interestingly, the inspection of this high resolution structure demonstrates that only minor alterations occur. Nevertheless, experiments indicate that Gly52 replacements in TmArgBP produce destabilizations comparable to those observed upon protein truncation or dissection in domains. Notably, we show that force-fields commonly used in computational biology do not reproduce this non-canonical state. Using TmArgBP as model system we here demonstrate that the structural context may force residues to adopt conformations believed to be strictly forbidden and that barely detectable alterations produce major destabilizations. Present findings highlight the role of subtle strains in governing protein stability. A full understanding of these phenomena is essential for an exhaustive comprehension of the factors regulating protein structures.

Counteraction of denaturant-induced protein unfolding is a general property of stabilizing agents.

DSC measurements on RNase A at neutral pH show that five stabilizing agents, namely trimethylamine N-oxide, glucose, sucrose, betaine and sodium sulfate, can counteract the destabilizing action of urea, sodium perchlorate, guanidinium chloride and guanidinium thiocyanate. This is an important finding inferring that counteraction has a common physical origin, regardless of the chemical differences among the stabilizing agents and among the destabilizing ones. A rationalization is provided grounded on the following line of reasoning: (a) the decrease in solvent-excluded volume effect is the main stabilizing contribution of the native state; (b) its magnitude increases on increasing the density of the aqueous solution; (c) the density increases significantly in the ternary solutions containing water, a stabilizing agent and a destabilizing one, as indicated by the present experimental data.

Domain communication in Thermotoga maritima Arginine Binding Protein unraveled through protein dissection.

Substrate binding proteins represent a large protein family that plays fundamental roles in selective transportation of metabolites across membrane. The function of these proteins relies on the relative motions of their two domains. Insights into domain communication in this class of proteins have been here collected using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model system. TmArgBP was dissected into two domains (D1 and D2) that were exhaustively characterized using a repertoire of different experimental and computational techniques. Indeed, stability, crystalline structure, ability to recognize the arginine substrate, and dynamics of the two individual domains have been here studied. Present data demonstrate that, although in the parent protein both D1 and D2 cooperate for the arginine anchoring; only D1 is intrinsically able to bind the substrate. The implications of this finding on the mechanism of arginine binding and release by TmArgBP have been discussed. Interestingly, both D1 and D2 retain the remarkable thermal/chemical stability of the parent protein. The analysis of the structural and dynamic properties of TmArgBP and of the individual domains highlights possible routes of domain communication. Finally, this study generated two interesting molecular tools, the two stable isolated domains that could be used in future investigations.