Molten Globule Research Articles

Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues.

The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as "Synechocystis hemoglobin (SynHb)". The "three histidines" linkages to heme are novel to this cyanobacterial hemoglobin. Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a "molten globule" like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability. Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.

Comparative study of the protein denaturing ability of different organic cosolvents

The influence of a wide spectrum of water-miscible organic cosolvents at different concentrations on the denaturation of hen egg-white lysozyme is studied using differential scanning calorimetry (DSC) and circular dichroism (CD). The denaturing ability of cosolvents is characterized with the parameter −∂Td∂x1 reflecting the change in the denaturation temperature with increasing cosolvent concentration. A series of cosolvents according to their denaturing ability is established: glycerol < ethylene glycol < pure water < dimethyl sulfoxide < methanol < ethanol < formamide < acetonitrile, dimethyl formamide, acetone < 2-propanol < 1,4-dioxane < tert-butanol < 1-propanol < tetrahydrofuran < 2-butanol < 1-butanol. The link of the −∂Td∂x1 parameter to the m values obtained in isothermal studies of chemically induced denaturation and to the solvation properties of aqueous-organic mixtures is demonstrated. Near-UV CD measurements indicate that changes in the tertiary structure occur at slightly lower temperature than the DSC peak in some of the mixtures with high organic cosolvent content. Far-UV CD measurements in the mixtures containing alcohols or tetrahydrofuran confirm non-simultaneous disruption of the tertiary and secondary lysozyme structure. Organic cosolvents induce formation of the molten globule state with preserved and even increased secondary structure, which gradually disrupts at higher temperatures.

Fluorescence quenching by ionic liquid as a potent tool to study protein unfolding intermediates

This study aimed to utilize the fluorescence quenching property of imidazolium-based ionic liquid to characterize the unfolding intermediates of proteins along the denaturation pathway. Low concentrations (<10 mM) of imidazolium-based ionic liquid are known to quench tryptophan fluorescence without affecting the protein structure; we have successfully utilized this property to evaluate the tertiary structure of intermediates in the GdmCl-induced denaturation pathway of bovine serum albumin (BSA). BSA is a globular protein with 583 amino acids and two intrinsic tryptophans. The Trp-fluorescence quenching experiments have been supplemented by measurements of Trp fluorescence lifetime, energy transfer efficiency between tryptophan and a nonpolar acceptor, 1,6-diphenyl-1,3,5-hexatriene (DPH), near- and far-UV circular dichroism measurements. Overall, our results revealed the denaturation of BSA to proceed via two parallel pathways in presence of low concentrations of GdmCl (≤1.5 M), wherein a dry molten globule was in equilibrium with the native conformation. At higher GdmCl concentrations, both the conformations entered the unfolding pathway, assuming a partially unfolded intermediate at 1.85 M GdmCl, which subsequently unfolded to form the denatured protein at yet higher GdmCl concentrations. Based on the fluorescence quenching property of imidazolium-based ionic liquid, we proposed a plausible mechanism of GdmCl-induced unfolding of BSA, which is in conformance with previous reports, although with more details. Therefore, the study suggested the potential of imidazolium-based ionic liquid as a reliable tool to investigate the intermediate conformations of BSA in the GdmCl-induced denaturation pathway, and proposed a mechanism of unfolding of BSA.

SUN-219 Electron Transport Chain Complex 2 in Mitochondrial Pregnenolone Synthesis

The mitochondrial P450 family of enzymes (SCC), which require the electron transport chain (ETC) complexes III, IV and V, initiate steroidogenesis by cleaving the sidechain of cholesterol to synthesize steroid hormones, an essential component for mammalian survival. SCC is required for full-term gestation, and aberrant expression may cause pseudohermaphroditism, breast cancer or polycystic ovary syndrome. Complex II or succinate dehydrogenase (quinone) is shared with the TCA cycle and has no proton pumping capacity and no known role in steroid synthesis. We now show that succinate is an intermediate metabolite in the TCA cycle and plays a central role physiologically. Specifically, complex II is required for SCC activation, where the proton pump facilitates an active intermediate state conformation at the matrix, so that in the presence of succinate, ATP can add phosphate. A longer intermediate equilibrium state generates a transient stabilization to enhance the binding of phosphate anions in the presence of succinate anions, resulting in higher enthalpy and activity. An inhibition of the processing at the intermediate state stops phosphate addition and activity. We further describe that phosphate circulation brings the molten globule, an intermediate, to an active folded state. This is the first report showing that an intermediate state activated by succinate facilitates ETC complex II interaction with complexes III and IV for metabolism.

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

The Molten Globule, and Two-State vs. Non-Two-State Folding of Globular Proteins.

From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.

The effect of extreme acid-induced soybean glycinin molten globules state on foaming ability

The molten globule (MG) state of soybean glycinin (11S), which is an unfolding quasi-intermediate state, has been achieved in an extremely acidic pH range (pH 0.5–3.0). By endogenous fluorescence, near and far ultraviolet circular dichroism, it was found that the secondary structure of soybean 11S protein was retained but the tertiary structure was stretched at pH 1.0. The1-anilino-8-naphthalene sulfonate (ANS) fluorescence spectrum and particle size measurement revealed that soybean 11S protein at pH 1.0 had greater hydrophobicity, and the particle size was between native state and fully denatured state. Taken together, the results show that soybean 11S protein could exist in a molten globule state at pH 1.0. The foaming characteristics of this state were analyzed according to foaming overrun and the foam microstructure. Relative to the native state, the foaming properties and foaming stability of MG-11S at pH 1.0 were significantly improved by 3.4 times and 3.1 times, respectively, and the mean life index of foam was significantly improved and the sizes of the bubbles generated were smaller by a polarizing microscope. Moreover, the zeta potential results show that the MG-11S system was relatively stable, with greater viscosity and lower surface tension. This study advances the understanding of the acid-induced MG state of soybean 11S protein and provides a theoretical basis to produce proteins with excellent foaming properties, which is helpful for applications in the food, biomedical and non-food fields.

Differential effect of biophenols on attenuation of AGE-induced hemoglobin aggregation

Despite most studied activities of natural biophenols rely on antioxidant properties, little clues explored their key structural components with regard to opposing action on glycation-induced aggregation. Herein, human hemoglobin (hHb)/fructose system used to decipher if structural peculiarities of two biophenols “chlorogenic acid (CGA) and curcumin (CUR)” are effective toward AGEs-bridged aggregate formation. Suppression in amyloid cross-β formation was monitored by CD spectroscopy, fluorescence microscopy, ANS and AGE fluorescence. Reduction in molten globule structure of modified-Hb by CGA was corroborated with helix structure, thiol group and lysine residues content estimation for native, glycated and biophenols treated samples. ThT and Congo red assays showed the cross-β breaking properties of CGA. Molecular docking outcomes revealed the positioning of CGA/CUR is driven by “aromatic interactions” with Trp β1180 and Tyr α2540. These interactions are modulated by the structural constraints such as number of hydroxyl groups and their methylation status directing the biophenols to the amyloidogenic core. The results are applicable to formulation of small-molecule nutraceuticals for treatment of conformational diseases.

How A Novel Scientific Concept Was Coined the "Molten Globule State".

As a tribute to Professor Oleg B. Ptitsyn, we organized an interview with Professor Akiyoshi Wada held in Tokyo in the middle of September 2019. Both Professor A. Wada and the late Professor O. B. Ptitsyn greatly contributed to the field of protein biophysics, and they played leading roles in establishing the concept of the “Molten Globule state” 35–40 years ago. This editorial is intended to recount, as accurately as possible, some episodes during the early days of protein research that led to the discovery of this state, and how this concept was coined the “Molten Globule state” and came to be widely accepted by biophysicists, biochemists, and molecular biologists.

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.

Structural and conformational behavior of MurE ligase from Salmonella enterica serovar Typhi at different temperature and pH conditions

MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.

Molten Globule Driven Liquid-Liquid Phase Separation at the Center of Viral Factory Assembly

Dynamic spatiotemporal distribution of biomolecules that share a biochemical path within cells takes place through liquid-liquid phase separation (LLPS) driven biomolecular condensates. “Viral factories” are liquid-like structures within the cytosol of infected cells and sites for transcription and replication. The respiratory syncytial virus (RSV) is a member of the mononegavirales order which include several serious pathogens. The replication complexes of these viruses consist of an RNA polymerase, L, the nucleoprotein N, which wraps the viral genome, and a phosphoprotein, P.

Transition between protein-like and polymer-like dynamic behavior: Internal friction in unfolded apomyoglobin depends on denaturing conditions

Equilibrium dynamics of different folding intermediates and denatured states is strongly connected to the exploration of the conformational space on the nanosecond time scale and might have implications in understanding protein folding. For the first time, the same protein system apomyoglobin has been investigated using neutron spin-echo spectroscopy in different states: native-like, partially folded (molten globule) and completely unfolded, following two different unfolding paths: using acid or guanidinium chloride (GdmCl). While the internal dynamics of the native-like state can be understood using normal mode analysis based on high resolution structural information of myoglobin, for the unfolded and even for the molten globule states, models from polymer science are employed. The Zimm model accurately describes the slowly-relaxing, expanded GdmCl-denaturated state, ignoring the individuality of the different aminoacid side chain. The dynamics of the acid unfolded and molten globule state are similar in the framework of the Zimm model with internal friction, where the chains still interact and hinder each other: the first Zimm relaxation time is as large as the internal friction time. Transient formation of secondary structure elements in the acid unfolded and presence of α-helices in the molten globule state lead to internal friction to a similar extent.

Effect of Green Synthesis of Gold Nanoparticles (AuNPs) from Hibiscus sabdariffa on the Aggregation of α-Lactalbumin

Amyloidosis are a hereditary or acquired disorders, which are associated with misfolding of normally soluble, functional peptides and proteins, and their conversion into contentious aggregation can cause amyloid fibrils. These fibrils reduce function of the proteins and with generation of toxic intermediates in the process of self-assembly cause amyloid diseases such as Alzheimer, Parkinson, etc. Nanoparticles are materials with a dimension of 1–100 nm that influence the process of protein fibrillation since nanoparticles mediated by chemical methods have been show side effects, biological methods have been preferred due to its safety for human therapeutic use. In this study, the effect of gold nanoparticle produced from hibiscus on the amyloid formation of α-lactalbumin was evaluated using light scattering absorption, fluorescence (ThT assay, intrinsic fluorescence assay and ANS binding assay), TEM, CD spectroscopy and SDS-PAGE. α -Lactalbumin has been chose as a good sample for study the amyloid formation due to the formation of molten globule state. The results show that AuNPs inhibit formation of amyloid fibrils in reduced α-lactalbumin. This protective effect resulted are due to increase protein adsorption to the surface of the nanoparticles and therefore prevents their structural changes. Nanoparticle by binding to the monomer prevented them from join together and elongation the core of amyloid fibrils. Finally, the results show that biosynthesized AuNPs mediated by hibiscus can be used as effective therapeutic agent for prevention of formation amyloid and treatment of amyloid disease.

Mimicking cotranslational folding of prosubtilisin E in vitro.

Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

Formation of molten globule state in horse heart cytochrome c under physiological conditions: Importance of soft interactions and spectroscopic approach in crowded milieu

To understand protein folding problem under physiological condition, usually taken as dilute aqueous buffer at pH 7.0 and 25 °C, knowledge of properties of folding intermediates is important, such as molten globule (MG). We observed that polyethylene glycol 400 Da (PEG 400) induces molten globule state conformation in cytochrome c at pH 7.0 and 25 °C. This PEG-induced MG state has: (i) native tertiary structure partially perturbed, (ii) unperturbed native secondary structure, (iii) newly exposed hydrophobic patches, and (iv) has 1.58 times more hydrodynamic volume than that of the native protein. Isothermal titration calorimetry and docking studies showed specific binding between PEG 400 and cytochrome c. The study delineates that PEG-protein interactions are more complex than the excluded-volume. The soft interactions need to be seriously studied in crowding milieu that leads to destabilization of protein and overcome stabilizing exclusion volume effect. This study not only can help in unraveling the mystery of steps involved in the proper folding of proteins to solve the massively complicated problems of protein folding but also provides novel insights towards importance of structural change in proteins inside cell where intermediate states of protein import-export easily via membranes rather than native form of proteins.

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

Segmental Motions of Proteins under Non-native States Evaluated Using Quasielastic Neutron Scattering.

Characterization of the dynamics of disordered polypeptide chains is required to elucidate the behavior of intrinsically disordered proteins and proteins under non-native states related to the folding process. Here we develop a method using quasielastic neutron scattering, combined with small-angle X-ray scattering and dynamic light scattering, to evaluate segmental motions of proteins as well as diffusion of the entire molecules and local side-chain motions. We apply this method to RNase A under the unfolded and molten-globule (MG) states. The diffusion coefficients arising from the segmental motions are evaluated and found to be different between the unfolded and MG states. The values obtained here are consistent with those obtained using the fluorescence-based techniques. These results demonstrate not only feasibility of this method but also usefulness to characterize the behavior of proteins under various disordered states.

Backbone and side chain 1H, 15N and 13C chemical shift assignments of the molten globule state of L94G mutant of horse cytochrome-c.

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25°C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25°C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments.

Ultra-Rapid Glutathionylation of Ribonuclease: Is this the Real Incipit of its Oxidative Folding?

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40–50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5’-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease.