Native State Of Globular Proteins Research Articles

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.

A quantum chemical study on the hydration energetics of trimethylamine N-oxide, its protonated form and tert-butanol

Trimethylamine N-oxide (TMAO) stabilizes the native state of globular proteins and counteracts the destabilizing action of denaturants. However, at pH values lower than its pKa = 4.7, TMAO destabilizes the native state. We studied, via DFT calculations, the interactions of TMAO, its protonated form and isosteric tert-butanol with one, two and three water molecules in vacuo, PCM and SMD water. The energetic strength of the H-bonds the considered solutes make with three water molecules, coupled to a model of globular protein stability, allows us to propose a rationalization of the pH-dependent TMAO behavior toward globular proteins.

The magnitude of macromolecular crowding caused by Dextran and Ficoll for the conformational stability of globular proteins

The polysaccharides Dextran and Ficoll are commonly used as crowding agents to mimic the cellular environment. They usually stabilize the native state of globular proteins, and this effect has been explained by the excluded volume idea: the reduction of the volume available to host the protein molecule shifts the equilibrium towards the folded conformation. However, the experimentally measured stabilization is lower than expected from theoretical models. We would like to show that, applying a classic scaled particle theory approach that considers the density of the solutions, we can provide reliable estimates of their stabilizing effect. Density is a fundamental requirement because it reflects the actual interactions among the solution molecules and allows the calculation of the number density and the volume packing density of the solution, two factors that play a major role in defining the magnitude of the solvent-excluded volume effect. Application of the model to aqueous solutions of glucose, sucrose, Dextran-70 and Ficoll-70 indicates that the stabilization effect of the two polysaccharides is similar to that of glucose and sucrose, and this happens due to the low number density of the polymer solutions.

Effect of heavy water on the conformational stability of globular proteins.

It is well established from the experimental point of view that the native state of globular proteins is more stable in heavy water than in water. No robust explanation, however, has been provided up to now. The application of the theoretical approach, originally devised to rationalize the general occurrence of cold denaturation, indicates that the magnitude of the solvent-excluded volume effect is slightly smaller in heavy water than in water and cannot explain the observed protein stabilization. The latter has to be due to the strength of protein-water van der Waals attractions which are weaker in heavy water due to the smaller molecular polarizability of D2 O compared with that of H2 O molecules. Since protein-water van der Waals attractions occur more in the denatured than in the native state, this contribution leads to a stabilization of the latter through a destabilization of the former.

Why does TMAO stabilize the globule state of PNIPAM?

Experimental measurements have shown that trimethylamine N-oxide, TMAO, is able to stabilize the globule state of PNIPAM [Phys.Chem.Chem.Phys. 2016; 18:31459], the same effect it has towards the native state of globular proteins. The TMAO stabilizing action is rationalized by means of the same theoretical approach used to address the entropic driving force and the effect of sodium salts, urea and tetramethylurea on PNIPAM collapse [Phys.Chem.Chem.Phys. 2015; 17:27750; Phys.Chem.Chem.Phys. 2016; 18:14426]. TMAO stabilizes the PNIPAM globule state because its addition to water causes an increase in the magnitude of the solvent-excluded volume effect, and only a small increase in the magnitude of the PNIPAM-solvent energetic attractions, since TMAO interacts preferentially with water.

Computational investigation of cold denaturation in the Trp-cage miniprotein

The functional native states of globular proteins become unstable at low temperatures, resulting in cold unfolding and impairment of normal biological function. Fundamental understanding of this phenomenon is essential to rationalizing the evolution of freeze-tolerant organisms and developing improved strategies for long-term preservation of biological materials. We present fully atomistic simulations of cold denaturation of an α-helical protein, the widely studied Trp-cage miniprotein. In contrast to the significant destabilization of the folded structure at high temperatures, Trp-cage cold denatures at 210 K into a compact, partially folded state; major elements of the secondary structure, including the α-helix, are conserved, but the salt bridge between aspartic acid and arginine is lost. The stability of Trp-cage's α-helix at low temperatures suggests a possible evolutionary explanation for the prevalence of such structures in antifreeze peptides produced by cold-weather species, such as Arctic char. Although the 310-helix is observed at cold conditions, its position is shifted toward Trp-cage's C-terminus. This shift is accompanied by intrusion of water into Trp-cage's interior and the hydration of buried hydrophobic residues. However, our calculations also show that the dominant contribution to the favorable energetics of low-temperature unfolding of Trp-cage comes from the hydration of hydrophilic residues.

Structure and Dynamics of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are involved in a wide range of essential biological processes, including in particular signalling and regulation. We are only beginning, however, to develop a detailed knowledge of the structure and dynamics of these proteins. It is becoming increasingly clear that, as IDPs populate highly heterogeneous states, they should be described in terms of conformational ensembles rather than as individual structures, as is instead most often the case for the native states of globular proteins. Within this context, in this chapter we describe the conceptual tools and methodological aspects associated with the description of the structure and dynamics of IDPs in terms of conformational ensembles. A major emphasis is given to methods in which molecular simulations are used in combination with experimental nuclear magnetic resonance (NMR) measurements, as they are emerging as a powerful route to achieve an accurate determination of the conformational properties of IDPs.

Why Do Tetrapropylammonium Chloride and Sulphate Salts Destabilize the Native State of Globular Proteins?

It has recently been shown that aqueous solutions of tetrapropylammonium chloride and sulphate salts destabilize the folded conformation of Trp-peptides (Dempsey et al., 2011). This result is rationalized by the application of a statistical thermodynamic approach (Graziano, 2010). It is shown that the magnitude of the solvent-excluded volume effect, the main contribution for the native state stability, decreases in both aqueous 2 M TPACl solution and aqueous 1 M TPA2SO4 solution. This happens because TPA+ ions are so large in size and interact so weakly with water molecules, due to their very low charge density, to be able to counteract the electrostrictive effect of chloride and sulphate ions on the water structure, so that the density of their aqueous solutions is smaller or only slightly larger than that of water.

Temperature and Urea Have Opposing Impacts on Polyproline II Conformational Bias

The native states of globular proteins have been accessed in atomic detail by X-ray crystallography and nuclear magnetic resonance spectroscopy, yet characterization of denatured proteins beyond global metrics has proven to be elusive. Denatured proteins have been observed to exhibit global geometric properties of a random coil polymer. However, this does not preclude the existence of nonrandom, local conformational bias that may be significant for protein folding and function. Indeed, circular dichroism (CD) spectroscopy and other methods have suggested that the denatured state contains considerable local bias to the polyproline II (PII) conformation. Here, we develop predictive models to determine the extent that temperature and the chemical denaturant urea modulate PII propensity. In agreement with our predictive model, PII propensity is observed experimentally to decrease with an increase in temperature. Conversely, urea appears to promote the PII conformation as determined by CD and isothermal titration calorimetry. Importantly, the calorimetric data are in quantitative agreement with a model that predicts the stability of the PII helix relative to other denatured state conformations based upon solvent accessible surface area and experimentally measured Gibbs transfer free energies. The ability of urea to promote the PII conformation can be attributed to the favorable interaction of urea with the peptide backbone. Thus, perturbing denatured states by temperature or cosolutes has subtle, yet opposing, impacts on local PII conformational biases. These results have implications for protein folding as well as for the function of signaling proteins that bind proline-rich targets in globular or intrinsically disordered proteins.

Subtle Monte Carlo Updates in Dense Molecular Systems.

Although Markov chain Monte Carlo (MC) simulation is a potentially powerful approach for exploring conformational space, it has been unable to compete with molecular dynamics (MD) in the analysis of high density structural states, such as the native state of globular proteins. Here, we introduce a kinetic algorithm, CRISP, that greatly enhances the sampling efficiency in all-atom MC simulations of dense systems. The algorithm is based on an exact analytical solution to the classic chain-closure problem, making it possible to express the interdependencies among degrees of freedom in the molecule as correlations in a multivariate Gaussian distribution. We demonstrate that our method reproduces structural variation in proteins with greater efficiency than current state-of-the-art Monte Carlo methods and has real-time simulation performance on par with molecular dynamics simulations. The presented results suggest our method as a valuable tool in the study of molecules in atomic detail, offering a potential alternative to molecular dynamics for probing long time-scale conformational transitions.

How does sucrose stabilize the native state of globular proteins?

It is well known that sucrose stabilizes the native state of globular proteins against both chemical denaturants and temperature. A largely accepted explanation of sucrose-induced stabilization is not yet emerged. It is shown that the same theoretical approach able to rationalize the occurrence of cold denaturation, the contrasting role of GdmCl and Gdm2SO4, and the TMAO counteraction of urea denaturing activity [PCCP 12 (2010) 14245; PCCP 13 (2011) 12008; PCCP 13 (2011) 17689] works well also in the case of sucrose. The solvent-excluded volume effect plays the fundamental role because sucrose addition to water causes a marked increase in volume packing density due to the large size of sucrose molecules, that act as crowding agents.

Molecular size scaling in families of protein native folds

The mean size of the most compact native states of globular proteins, independent of folding type, follows the scaling law of collapsed polymers Rg ~ n1/3, relating the radius of gyration Rg to the number of protein residues, n. Until now, this behaviour has only been observed within a small subset of unrelated single-domain proteins with n < 300. Here, we employ the SCOP database of protein folds to study systematically the scaling behaviour of well-defined families of domains that share structural and functional characteristics. In the particular case of helical proteins, we identify the folding types that can be associated with scaling laws corresponding to compact behaviour (e.g., the cytochrome-C monodomains) and noncompact behaviour (e.g., the immunoglobulin/albumin-binding and spectrin-repeat domains). Our results quantify the size variations within some folding families, as well as reveal that some distinct folds represent structures with equivalent compactness.

On the cold denaturation of globular proteins

The recent finding that yeast frataxin shows, at pH 7.0, cold denaturation at 274 K and hot denaturation at 303 K [A. Pastore, S.R. Martin, A. Politou, K.C. Kondapalli, T. Stemmler, P.A. Temussi, J. Am. Chem. Soc. 129 (2007) 5374] calls for a deeper rationalization of the molecular mechanisms stabilizing–destabilizing the native state of globular proteins. It is shown that the statistical thermodynamic model originally developed by Ikegami can reproduce in a more-than-qualitative manner the two conformational transitions of yeast frataxin, providing important clues on their molecular origin.

Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

In this paper we use the results from all-atom molecular dynamics (MD) simulations of proteins and peptides to assess the individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the "unfolded state") and charged amino acids in globular proteins (the "folded state"). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO(-)) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while N(+)(3) groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously.

Characterizing residual structure in disordered protein States using nuclear magnetic resonance.

The importance of disordered protein states in biology is gaining recognition, and can be attributed in part to the participation of unfolded and partially folded states of globular proteins in normal and abnormal biological functions, such as protein translation, protein translocation, protein degradation, protein assembly, and protein aggregation (1-5). There is also a growing awareness that a significant fraction of gene products from various genomes, including the human genome, fall into a category that includes low complexity, low globularity, or intrinsically unstructured proteins (6-9). Unlike native states of globular proteins, disordered protein states, by definition, do not adopt a fixed structure that can be determined using classical high-resolution methods. Nevertheless, there has long been evidence that many disordered states contain detectable and significant residual or nascent structure (10-16). This structure has been found to be important for nucleating local structure, as well as mediating long range contacts upon either intramolecular folding to the native state (17-21) or intermolecular folding with specific binding partners (22-24), and is also predicted to influence intermolecular folding into structured aggregates (25,26). The primary tool for the characterization of such structure is high-resolution solution state nuclear magnetic resonance (NMR) spectroscopy. Advances in NMR instrumentation and methods have greatly facilitated this task and in principle can now be accomplished by those without extensive prior experience in NMR spectroscopy. This chapter describes how this can be accomplished.

Analytical estimation of scaling behavior for the entanglement complexity of a bond network.

Geometrical entanglements in a polymer network can be characterized in terms of the mean number of projected bond-bond crossings, N. Here, we present an analytical method to study the dependence of N on the number of bonds in the network, n. Our approach shows the occurrence of power-law scaling, N - n(beta). The estimated upper bound to the exponent for maximally compact networks, Beta approximately 1.38, agrees well with the values observed in simulations of transient networks in liquids and in the folding features of native states of globular proteins.

Probability of triple contacts in polymer chains

The compact conformations of polymers are important because they are the principal configurations of the native states of globular proteins. The probability distributions and the correlation functions of triple contacts are calculated using the Gaussian statistics of the random-flight chain. Some comparisons with single and double contacts are also made.

Role of hydrophobic interactions on the stabilisation of native state of globular proteins

The technique of intensity photon correlation spectroscopy has been utilised to investigate the native conformation of lysozyme in water/ethanol mixture as a function of alcohol concentration in the water-rich region of composition (cosolvent mole fraction x 2<0.08). A non-trivial behaviour of the hydrodynamic radius is obtained, characterised by a minimum at x 2=0.02 and a maximum at x 2=0.06. This behaviour is similar to that of partial molar volume of ethanol in water and reflects changes in the alcohol/water structure. The results are discussed in connection to the effect of alcohol in modulating solvent-mediated interactions.

Mutational analysis of the propensity for amyloid formation by a globular protein

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.

Artificial chaperoning of insulin, human carbonic anhydrase and hen egg lysozyme using linear dextrin chains – a sweet route to the native state of globular proteins

Linear dextrins (α-1,4- d-glucopyranoside chains) are known to possess amphiphilic surfaces and solubilize lipophilic compounds. We have assessed the ability of this amphiphilic surface of dextrin to inhibit the self-aggregation and assist the refolding of proteins. Addition of decameric dextrin, or dextrin-10, in the renaturation buffer improves the refolding yield of human carbonic anhydrase from its guanidinium chloride-induced denatured state. It is also seen to inhibit the self-aggregation of insulin. The ability of dextrin-10 to interact with cetyltrimethylammonium bromide and postpone its critical micellar concentration allows the use of dextrin-10 as a `detergent stripping agent' in a novel artificial chaperoning process described earlier. The aggregation of human carbonic anhydrase and lysozyme upon refolding is prevented by cetyltrimethylammonium bromide due to the formation of a protein-detergent complex; dextrin-10 strips off the detergent from the complex and allow the proteins to fold, thus increasing the renaturation yield. Dextran-4 (the α-1,6- d-glucopyranoside chain), which does not exhibit amphiphilic properties, does not help in such artificial chaperoning.