Finite-size effects of Kirkwood–Buff integrals from molecular simulations

The modelling of thermodynamic properties of liquids from local density fluctuations is relevant to many chemical and biological processes. The Kirkwood–Buff (KB) theory connects the microscopic structure of isotropic liquids with macroscopic properties such as partial derivatives of activity coefficients, partial molar volumes and compressibilities. Originally, KB integrals were formulated for open and infinite systems which are difficult to access with standard Molecular Dynamics (MD) simulations. Recently, KB integrals for finite and open systems were formulated (J Phys Chem Lett. 2013;4:235). From the scaling of KB integrals for finite subvolumes, embedded in larger reservoirs, with the inverse of the size of these subvolumes, estimates for KB integrals in the thermodynamic limit are obtained. Two system size effects are observed in MD simulations: (1) effects due to the size of the simulation box and the size of the finite subvolume embedded in the simulation box, and (2) effects due to computing radial distribution functions (RDF) from a closed and finite system. In this study, we investigate the two effects in detail by computing KB integrals using the following methods: (1) Monte Carlo simulations of finite subvolumes of a liquid with an analytic RDF and (2) MD simulations of a WCA mixture for various simulation box sizes, but at the same thermodynamic state. We investigate the effect of the size of the simulation box and quantify the differences compared to KB integrals computed in the thermodynamic limit. We demonstrate that calculations of KB integrals should not be extended beyond half the size of the simulation box. For finite-size effects related to the RDF, we find that the Van der Vegt correction (J Chem Theory Comput. 2013;9:1347) yields the most accurate results.     

A comparison of force fields for ethanol–water mixtures

Aqueous ethanol mixtures are studied through molecular dynamics simulations with the focus on exploring how various force field models reproduce the association and its influence on selected thermo-physical properties of these mixtures. The most important conclusion seems to be the inadequacy of all classical force fields to reproduce the very peculiar shape of the excess enthalpy of these mixtures, as a function of the ethanol concentration, neither quantitatively nor qualitatively. The Kirkwood–Buff (KB) integrals calculated using the simulation data follow the same trends as the experimental ones. This suggests complicated correlation of the excess enthalpy with the concentration fluctuation and clustering in these mixtures. The KB force field shows better overall agreement with experimental results than the other studied models.     

Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

Living with urea stress

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments. These osmolytes protect intracellular macromolecules against denaturing environmental stress. In contrast to the usually benign effects of most organic osmolytes, the waste product urea is a well-known perturbant of macromolecules. Although urea is a perturbing solute which inhibits enzyme activity and stability, it is employed by some species as a major osmolyte. The answer to this paradox was believed to be the discovery of protective osmolytes (methylamines). We review the current state of knowledge on the various ways of counteracting the harmful effects of urea in nature and the mechanisms for this. This review ends with the mechanistic idea that cellular salt (KCl/NaCl) plays a crucial role in counteracting the effects of urea, either by inducing required chaperones or methylamines, or by thermodynamic interactions with urea-destabilised proteins. We also propose future opportunities and challenges in the field.     

Strategies for Correcting the AF508 CFTR Protein-Folding Defect

Many human diseases arise as a result of mutations within genes encoding essential proteins. In many cases, the mutations are not so severe as to render the protein biologically inactive. Rather, the mutations oftentimes result in only subtle protein-folding abnormalities. In the case of the CFTR protein, a mutation leading to the loss of a single amino acid is responsible for the diseased state in the majority of individuals with cystic fibrosis. Here the newly synthesized mutant CFTR protein, missing a phenylalanine residue at position 508 (F508 CFTR), is unable to transit from the endoplasmic reticulum to the plasma membrane, where it functions as a regulator of chloride transport. All of the available evidence indicate that the newly synthesized F508 CFTR protein adopts a slightly altered conformation and therefore is retained at the level of the endoplasmic reticulum, ostensibly by the actions of the cellular quality control system. Because the mutant protein is capable of functioning as a chloride channel, developing ways to elicit its release out of the ER and to the plasma membrane has important clinical implications. Herein, we discuss our recent studies showing that the protein folding defect associated with the F508 CFTR mutation, as well as a number of other temperature-sensitive mutations, can be overcome by strategies designed to influence protein folding inside the cell. Specifically we show that a number of low-molecular-weight compounds, all of which are known to stabilize proteins in their native conformation, are effective in rescuing the folding and/or processing defects associated with different mutations that oftentimes lead to human disease.     

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ(XP), contrary to conventional thinking that positive Γ(XP)'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ(XP), which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation.     

The therapeutic potential of chemical chaperones in protein folding diseases

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.     

Rapid collapse precedes the fast two-state folding of the cold shock protein

The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by F?rster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.     

Kinetic characterization of early intermediates in the folding of E. coli tryptophan-synthase beta 2 subunit

This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the beta 2 subunit of E. coli tryptophan-synthase. Two early folding steps are thus identified and characterized. One is very rapid (its rate constant at 12 degrees C is 0.02 sec-1) and corresponds to the folding of the N-terminal domain into a structure whose overall features approximate well those of the native domain. The second step is somewhat slower (its rate constant at 12 degrees C is 0.008 sec-1) and involves a conformational rearrangement of the N-terminal domain brought about by the interactions between the N- and C-terminal domains within a monomeric beta chain. This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric beta 2 subunit.