Folding of poly-amino acids and intrinsically disordered proteins in overcrowded milieu induced by pH change

pH-induced structural changes of the synthetic homopolypeptides poly-E, poly-K, poly-R, and intrinsically disordered proteins (IDPs) prothymosin alpha (ProT alpha) and linker histone H1, in concentrated PEG solutions simulating macromolecular crowding conditions within the membrane-less organelles, were characterized. The conformational transitions of the studied poly-amino acids in the concentrated PEG solutions depend on the polymerization degree of these homopolypeptides, the size of their side chains, the charge distribution of the side chains, and the crowding agent concentration. The results obtained for poly-amino acids are valid for IDPs having a significant total charge. The overcrowded conditions promote a significant increase in the cooperativity of the pH-induced coil-alpha-helix transition of ProTa and provoke histone H1 aggregation. The most favorable conditions for the pH-induced structural transitions in concentrated PEG solutions are realized when the charged residues are grouped in blocks, and when the distance between the end of the side group carrying charge and the backbone is small. Therefore, the block-wise distribution of charged residues within the IDPs not only plays an important role in the liquid-liquid phase transitions, but may also define the expressivity of structural transitions of these proteins in the overcrowded conditions of the membrane-less organelles. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Differences in the Pathways of Proteins Unfolding Induced by Urea and Guanidine Hydrochloride: Molten Globule State and Aggregates

It was shown that at low concentrations guanidine hydrochloride (GdnHCl) can cause aggregation of proteins in partially folded state and that fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates rather than with hydrophobic clusters on the surface of protein in molten globule state. That is why the increase in ANS fluorescence intensity is often recorded in the pathway of protein denaturation by GdnHCl, but not by urea. So what was previously believed to be the molten globule state in the pathway of protein denaturation by GdnHCl, in reality, for some proteins represents the aggregates of partially folded molecules

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters

Native Globular Actin Has a Thermodynamically Unstable Quasi-stationary Structure with Elements of Intrinsic Disorder

The native form of globular actin, G-actin, is formed in vivo as a result of complex post-translational folding processes that require ATP energy expenditure and are assisted by the 70 kDa heat shock protein, prefoldin and chaperonin containing TCP-1. G-actin is stabilized by the binding of one ATP molecule and one Ca2+ ion (or Mg2+ in vivo). Chemical denaturants, heating or Ca2+ removal transform native actin (N) into ‘inactivated actin’ (I), a compact oligomer comprising 14–16 subunits. Viscogenic and crowding agents slow this process but do not stop it. The lack of calcium in the solution accelerates the spontaneous N → I transition. Thus, native G-actin has a kinetically stable (as a result of the high free energy barrier between the N and I states) but thermodynamically unstable structure, which, in the absence of Ca2+ or other bivalent metal ions, spontaneously converts to the thermodynamically stable I state. It was noted that native actin has much in common with intrinsically disordered proteins: it has functionally important disordered regions; it is constantly in complex with one of its numerous partners; and it plays key roles in many cellular processes, in a manner similar to disordered hub proteins. By analyzing actin folding in vivo and unfolding in vitro, we advanced the hypothesis that proteins in a native state may have a thermodynamically unstable quasi-stationary structure. The kinetically stable native state of these proteins appears forcibly under the influence of intracellular folding machinery. The denaturation of such proteins is always irreversible because the inactivated state, for which the structure is determined by the amino acid sequence of a protein, comprises the thermodynamically stable state under physiological conditions

Actinous Enigma Or Enigmatic Actin: Folding, Structure, and Functions of the Most Abundant Eukaryotic Protein

Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins

Actin aggregation induced by GdnHCl in low concentrations.

<p>The dependence of ANS fluorescence intensity (<b>A</b>) and of light scattering (<b>B</b>) of the solutions of inactivated actin (red) and initially native actin after 10 min and 24 h of incubation in a solution of denaturants at the appropriate concentration (blue and green) on GdnHCl (closed symbols) and urea (open symbols) concentration. <b>Insert</b> in panel <b>A</b>. Scheme of actin denaturation and aggregation (N, U^* and I are native, essential unfolded and inactivated actin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015035#pone.0015035-Kuznetsova2" target="_blank">[11]</a>, I_ag is aggregates of inactivated actin, the details are given in the text). <b>Insert</b> in panel <b>B</b>. The dependence of the total macromolecule charge of actin on the pH of a solution calculated on the basis of protein amino acid content <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015035#pone.0015035-Toldo1" target="_blank">[24]</a>. The protein concentration was 0.15 mg/ml, ANS concentration was 5⋅10⁻⁵ M.</p

Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States

Protocol of determination of binding stoichiometry and affinity of fluorescent dyes with proteins in different structural states is proposed. The proposed approach is based on the spectrophotometric determination of concentrations of dye bound to protein and free dye in solutions prepared by equilibrium microdialysis. This technique allows also determining spectral properties of the bound dyes. The restrictions of the use of dye fluorescence intensity for characterization of its interaction with the target protein are discussed. It is shown that the dependence of the dye fluorescence intensity on its optical density together with the data on its binding parameter can give information about the dye fluorescence quantum yield. All procedures are illustrated by interaction of 8-anilino-1-naphthalenesulfonate (ANS) with bovine serum albumin

The Effects of Crowding Agents Dextran-70k and PEG-8k on Actin Structure and Unfolding Reaction

Recently, an increasing number of studies on proteins\u27 structure, stability and folding are trying to bring the experimental conditions closer to those existing in a living cell, namely to the conditions of macromolecular crowding. In vitro such conditions are typically imitated by the “inert” highly water-soluble polymers with different hydrodynamic dimensions. In this work, the effects of crowded milieu on the structure and conformational stability of actin, which is a key component of the muscle contraction system, was examined. The crowded milieu was simulated by high concentrations of PEG-8k or Dextran-70k. It was revealed that both crowding agents decelerated but not inhibited actin unfolding and made a compact state of inactivated actin thermodynamically more favorable in comparison with the unfolded state. At the same time, the high viscosity of the solution of crowding agents slowed down all processes and especially inactivated actin formation, since it involves the interaction of 14–16 partially unfolded actin molecules. The effects of crowding agent were larger when its hydrodynamic dimensions were closer to the size of globular actin