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Known crystal structures of 10 cytochromes (cyts) c from different sources led to the conclusion that natural mutations in these proteins does not affect their 3D structure, hence evolution preserved structure for function. A sequence alignment of horse cyt c with all other 284 cyts c led to two important conclusions: (i) Leu at position 94 is conserved in all 30 mammalian known sequences, and (ii) there are 14 other species which have either Val or Ile at 94th position. We asked a question: Is the avoidance of substitution by Val or Ile at position 94 in the mammalian cyts c by design or by chance? To answer this question, we introduced natural substitutes of Leu94 by Val and Ile in horse cyt c using site-directed mutagenesis. Here, from our in vitro and molecular dynamic simulation studies on L94V and L94I mutants, we concluded that (i) although the natural mutations destabilize the wild type cyt c, it does not significantly affect the mechanism of folding of the protein, (ii) urea-induced denaturation of WT cyt c and its mutants is a two-state process, and (iii) denaturation of WT cyt c and its mutants by guanidinium chloride is not a two-state process., Competing Interests: Declaration of competing interest Authors declare there are no any financial or commercial conflict of interests with the current article content., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Myoglobin is known to show formation of intermediate states under various environmental conditions, in spite of that, this is the first evidence of formation pre-molten globule (PMG) in myoglobin. Polyethylene glycol (PEG) of various molecular sizes shows assorted effects on different proteins. Out of too short and too long PEGs, only PEGs of optimal size interact with proteins leading to change in protein structure that form intermediate state. We are the first one to report the formation of PMG in a protein in the presence of a crowding agent. The PEG-induced intermediate state was characterized by various techniques like absorption, fluorescence, near- and far-UV circular dichroism spectroscopy, ANS binding, and dynamic light scattering measurements to be PMG. Isothermal titration calorimetry and docking studies were further carried out to delineate the mechanism of formation of PMG in myoglobin in physiological conditions. The intermediate formed due to interaction of PEG with myoglobin has physiological implications which are essential to unravel the mystery to solve the massively complicated problems involved in the proper folding of proteins in vivo. Further, outcomes from this study are expected to gain mechanistic insights on the native structure and functions of proteins under in vivo conditions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recently, we had published that urea-induced denaturation curves of optical properties of sheep serum albumin (SSA) are biphasic with a stable intermediate that has characteristics of molten globule (MG) state. In this study, we have extended the work by carrying out urea- and guanidinium chloride (GdmCl)-induced denaturations of SSA in the presence of naturally occurring mammalian kidney osmolytes, namely, sorbitol, myo-inositol and glycine betaine. We have observed that all these osmolytes (i) transform this biphasic transition into a co-operative, two-state transition and (ii) increase the stability of the protein in terms of midpoint of denaturation (C m ) and Gibbs free energy change in the absence of both denaturants (ΔG D 0 ). The relative effectiveness of different osmolytes on the stability of SSA follows the order: glycine betaine>myo-inositol>sorbitol. In this paper, we also report that kidney osmolytes destabilize MG state by shifting the equilibrium, native state↔MG state toward the left. This study will be helpful in understanding the existence of osmolytes in kidney and their role in folding of kidney proteins soaked with urea., (Copyright © 2017 Elsevier B.V. All rights reserved.)

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε400) and CD ([θ]409), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.

The conformational and functional state of biliproteins can be determined by optical properties of the covalently linked chromophores. α-Subunit of most of the phycoerythrin contains 164 residues. Recently determined crystal structure of the naturally truncated form of α-subunit of cyanobacterial phycoerythrin (Tr-αC-PE) lacks 31 N-terminal residues present in its full length form (FL-αC-PE). This provides an opportunity to investigate the structure-function relationship between these two natural forms. We measured guanidinium chloride (GdmCl)-induced denaturation curves of FL-αC-PE and Tr-αC-PE proteins, followed by observing changes in absorbance at 565nm, fluorescence at 350 and 573nm, and circular dichroism at 222nm. The denaturation curve of each protein was analyzed for ΔGD(∘), the value of Gibbs free energy change on denaturation (ΔGD) in the absence of GdmCl. The main conclusions of the this study are: (i) GdmCl-induced denaturation (native state↔denatured state) of FL-αC-PE and Tr-αC-PE is reversible and follows a two-state mechanism, (ii) FL-αC-PE is 1.4kcalmol(-1) more stable than Tr-αC-PE, (iii) truncation of 31-residue long fragment that contains two α-helices, does not alter the 3-D structure of the remaining protein polypeptide chain, protein-chromophore interaction, and (iv) amino acid sequence of Tr-αC-PE determines the functional structure of the phycoerythrin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

It is known from in vitro studies that macromolecular crowding in the cell effects protein structure, stability and function; but predictive studies are relatively unexplored. There are few reports where the effect of various crowder mixtures has been exploited to discern their combined effect on the structural stability of proteins. These studies are more significant because their effect can mimicked with in vivo conditions, where the environment is heterogeneous. Effects of two crowders, polyethylene glycol (PEG 400 Da), and its monomer ethylene glycol (EG) alone and in mixture on the structural stability of cytochrome c (cyt c) were determined using various spectroscopic and bioinformatics tools. The main conclusions of our study are (i) the monomer EG has a kosmotropic effect on the protein (stabilizes the protein), and has no significant effect on the tertiary structure; (ii) PEG 400 destabilizes the structure as well as the stability of the protein; and (iii) EG counteracts the destabilizing effect of PEG 400. From this investigation, it seems evident that proteins may fold or unfold in the crowded environment of the cell where various interactions assist them to maintain their structure for their functions. Bioinformatics approaches were also used to support all of the in vitro observations. Cyt c is functional protein; if the structure of the protein is modulated due to change in the environment its nature of function will also change. Our research addresses the question by modulating the environment around the protein, and the macromolecule (protein) conformation dynamics and interaction study via in vitro and in silico approaches which indirectly compares with that of the environment in-cellular milieu, which is highly crowded. [ABSTRACT FROM AUTHOR]

Proteins are indispensable to cellular communication and metabolism. The structure on which cells and tissues are developed is deciphered from proteins. To perform functions, proteins fold into a three-dimensional structural design, which is specific and fundamentally determined by their characteristic sequence of amino acids. Few of them have structural versatility, allowing them to adapt their shape to the task at hand. The intermediate states appear momentarily, while protein folds from denatured (D) ⇔ native (N), which plays significant roles in cellular functions. Prolific effort needs to be taken in characterizing these intermediate species if detected during the folding process. Protein folds into its native structure through definite pathways, which involve a limited number of transitory intermediates. Intermediates may be essential in protein folding pathways and assembly in some cases, as well as misfolding and aggregation folding pathways. These intermediate states help to understand the machinery of proper folding in proteins. In this review article, we highlight the various intermediate states observed and characterized so far under in vitro conditions. Moreover, the role and significance of intermediates in regulating the biological function of cells are discussed clearly. [ABSTRACT FROM AUTHOR]

Cytochrome c (cyt c) is widely used as a model protein to study (i) folding and stability aspects of the protein folding problem and (ii) structure–function relationship from the evolutionary point of view. Databases of cyts c now contain 285 cyt c sequences from different organisms. A sequence alignment of all these proteins with respect to horse cyt c led to several important conclusions. One of them is that Leu94 is always conserved in all 30 mammalian cyts c. It is known that mutation L94G of the wild type (WT) horse cyt c is destabilizing and mutant exists as molten globule under the native condition (buffer pH 6 and 25 °C). We have expressed and purified uniformly labeled (13C and 15N) and unlabeled WT horse cyt c and its L94G mutant. We report that labeling does not affect the thermodynamic stability of proteins. To support this conclusion, the secondary and tertiary structure of each protein in labeled and unlabeled forms was determined by conventional techniques (UV–Vis absorption and circular dichroism spectroscopy). [ABSTRACT FROM AUTHOR]

Oligonucleotide/oligosaccharide-binding (OB)-fold proteins play essential roles in the regulation of genome and its correct transformation to the subsequent generation. To maintain the genomic stability, OB-fold proteins are implicated in various cellular processes including DNA replication, DNA repair, cell cycle regulation and maintenance of telomere. The diverse functional spectrums of OB-fold proteins are mainly due to their involvement in protein–DNA and protein–protein complexes. Mutations and consequential structural alteration in the OB-fold proteins often lead to severe diseases. Here, we have investigated the structure, function and mode of action of OB-fold proteins (RPA, BRCA2, DNA ligases and SSBs1/2) in cellular pathways and their relationship with diseases and their possible use in therapeutic intervention. Due to the crucial role of OB-fold proteins in regulating the key physiological process, a detailed structural understanding in the context of underlying mechanism of action and cellular complexity offers a new avenue to target OB-proteins for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Hemochromatosis factor E (HFE) is a member of class I MHC family and plays a significant role in the iron homeostasis. Denaturation of HFE induced by guanidinium chloride (GdmCl) was measured by monitoring changes in [θ]222 (mean residue ellipticity at 222 nm), intrinsic fluorescence emission intensity at 346 nm (F346) and the difference absorption coefficient at 287 nm (Δϵ287) at pH 8.0 and 25°C. Coincidence of denaturation curves of these optical properties suggests that GdmClinduced denaturation (native (N) state ↔denatured (D) state) is a two-state process. The GdmCl-induced denaturation was found reversible in the entire concentration range of the denaturant. All denaturation curves were analyzed for ΔG0D , Gibbs free energy change associated with the denaturation equilibrium (N state ↔ D state) in the absence of GdmCl, which is a measure of HFE stability. We further performed molecular dynamics simulation for 40 ns to see the effect of GdmCl on the structural stability of HFE. A well defined correlation was established between in vitro and in silico studies. [ABSTRACT FROM AUTHOR]

There are a large number of biomolecules that are accountable for the extremely crowded intracellular environment, which is totally different from the dilute solutions, i.e., the idealized conditions. Such crowded environment due to the presence of macromolecules of different sizes, shapes, and composition governs the level of crowding inside a cell. Thus, we investigated the effect of different sizes and shapes of crowders (ficoll 70, dextran 70, and dextran 40), which are polysaccharide in nature, on the thermodynamic stability, structure, and functional activity of two model proteins using UV-Vis spectroscopy and circular dichroism techniques. We observed that (a) the extent of stabilization of α-lactalbumin and lysozyme increases with the increasing concentration of the crowding agents due to the excluded volume effect and the small-sized and rod-shaped crowder, i.e., dextran 40 resulted in greater stabilization of both proteins than dextran 70 and ficoll 70; (b) structure of both the proteins remains unperturbed; and (c) enzymatic activity of lysozyme decreases with the increasing concentration of the crowder. [ABSTRACT FROM AUTHOR]

Microtubule affinity regulating Kinase 4 (MARK4) belongs to the family of AMP-activated protein kinase. It phosphorylates microtubule associated proteins at specific sites (Serine in KXGS motifs) in the microtubule-binding repeats. In our previous studies, two constructs, namely, kinase domain with 59 N-terminal residues (residues 1–310) and only kinase domain (residues 59–310) of MARK4 show aggregation at physiological pH. However, these two constructs were stable at extremes of pH conditions. Now the question arises: how is MARK4 stable at physiological pH in-vivo ? To answer this question, we have successfully cloned, expressed, and purified UBA-domain along with the kinase domain of MARK4 and performed spectroscopic measurements and activity assays. We observed a pronounced secondary and tertiary structure and ATPase activity in the MARK4 at physiological pH. In conclusion, UBA domain may be important to maintain the structure, stability and activity of MARK4 under physiological conditions. [ABSTRACT FROM AUTHOR]

Abstract: We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine β-lactoglobulin A (β-lgA) at pH 2.0 and 25 °C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements. Following conclusions have been reached. (a) Urea-induced denaturation is not a two-state process. (b) GdmCl-induced denaturation is composed of two distinct two-state processes. (c) α-Helical content, burial of tryptophan residues and burial of hydrophobic surface area are more in the GdmCl-induced stable intermediate than those originally present in the native protein. [Copyright &y& Elsevier]

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 °C by observing changes in difference molar absorbance at 400 nm (Δϵ400), mean residue ellipticities at 222 nm ([θ]222) and difference mean residue ellipticity at 409 nm (Δ[θ]409). The denaturation is a three-step process when measured by Δϵ400 and Δ[θ]409, and it is a two-step process when monitored by [θ]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state ↔ MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions. [ABSTRACT FROM AUTHOR]

The interior of the cell is crowded with different kinds of biological molecules with varying sizes, shapes and compositions which may affect physiological processes especially protein folding, protein conformation and protein stability. To understand the consequences of such a crowded environment, pH-induced unfolding of holo alpha-lactalbumin (holo α-LA) was studied in the presence of ethylene glycol (EG). The effect of EG on the folding and stability of holo α-LA in aqueous solution was investigated using several spectroscopic techniques. The results indicate that stabilization/destabilization of holo α-LA by EG is concentration- and pH-dependent. Low concentration of EG stabilizes the protein at pH near its pI. From the results of far-UV CD, UV–visible and ANS fluorescence, intermediate state (MG state) was characterized in the presence of high concentration of ethylene glycol. The results invoke a new mechanism for the formation of MG state identical to active component of BAMLET. MG state of holo α-LA has a direct implication to cancer therapy. MG state of α-LA in complex with specific type of lipid is a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to the cancer cells. [ABSTRACT FROM AUTHOR]

The oligonucleotide/oligosaccharide-binding (OB)-fold domain proteins are considered as genome guardians, whose functions are extending beyond genomic stability. The broad functional diversity of the OB-fold proteins is attributed to their protein-DNA, protein-RNA, and protein-protein interactions (PPI). To understand the connectivity of the human OB-fold proteins, we report here a systems-level approach. Specifically, we mapped all human OB-fold PPI networks and evaluated topological features such as network robustness and network hub, among others. We found that the OB-fold network comprised of 227 nodes forming 5523 interactions, and has a scale-free topology having UBA52, ATR, and TP53 as leading hub proteins that control efficient communication within the network. Furthermore, four different clusters and subclusters have been identified, which are implicated in diverse cellular processes, including DNA replication, repair, maintenance of genomic stability, RNA processing, spermatogenesis, complement system, and telomere maintenance. The importance of these clusters is further strengthened by knockout studies, which showed a significant decrease in topological properties. In summary, this study provides new insights on the role of OB-fold protein as genome guardians in regard to the underlying mechanism of signaling pathways, the roles of key regulators, and thus, offers new prospects as potential targets for diagnostics and therapeutics purposes. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase kinase-3 (PDK3) plays important role in the glucose metabolism and is associated with cancer progression, and thus being considered as an attractive target for cancer therapy. In this study, we employed spectroscopic techniques to study the structural and conformational changes in the PDK3 at varying pH conditions ranging from pH 2.0 to 12.0. UV/Vis, fluorescence and circular dichroism spectroscopic measurements revealed that PDK3 maintains its native-like structure (both secondary and tertiary) in the alkaline conditions (pH 7.0–12.0). However, a significant loss in the structure was observed under acidic conditions (pH 2.0–6.0). The propensity of aggregate formation at pH 4.0 was estimated by thioflavin T fluorescence measurements. To further complement structural data, kinase activity assay was performed, and maximum activity of PDK3 was observed at pH 7.0–8.0 range; whereas, its activity was lost under acidic pH. To further see conformational changes at atomistic level we have performed all-atom molecular dynamics at different pH conditions for 150 ns. A well defined correlation was observed between experimental and computational studies. This work highlights the significance of structural dependence of pH for wide implications in protein-protein interaction, biological function and drug design procedures. • Effect of pH on the structure and function of PDK3 was investigated using both experimental and computational methods. • PDK3 shows best enzyme activity in the pH range of 7.0–8.0. • PDK3 maintains its structure and activity under alkaline pH range however, its activity was lost under acidic pH. • A well-defined correlation was observed in between spectroscopic and MD simulation studies. [ABSTRACT FROM AUTHOR]

Sphingosine kinase 1 (SphK1) is a lipid kinase which plays vital role in the regulation of varieties of biological processes including, cell growth, apoptosis and mitogenesis. In the present study, we investigated the guanidinium chloride (GdmCl)-induced denaturation of SphK1 at pH 8.0 and 25 °C using two different spectroscopic probes, i.e., mean residue ellipticity at 222 nm ([ θ ] 222) and fluorescence emission maxima (λ max). A significant overlap between the transition curves obtained from both the spectral properties indicate that GdmCl-induced unfolding of SphK1 follows two-state process i.e., Native (N) ⇌ Denatured (D) state. Interestingly, a visible protein aggregation was observed at low concentrations of GdmCl ([GdmCl] ≤ 1.5 M). The analysis of transition curves was done to estimate the thermodynamic parameters associated with the stability of SphK1. To complement our experimental findings, 100 ns molecular dynamics (MD) simulations were performed. Spectroscopic studies together with MD simulations provided mechanistic insights of unfolding pathway of SphK1 along with its stability parameters. Description: Normalized GdmCl-induced denaturation plot of SphK1 (left panel). FEL generated by projecting the principal components, PC1 and PC2, of SphK1 during MD simulations at 0 M and 6.0 M GdmCl. Unlabelled Image • SphK1 plays a crucial role in various biological processes including cell growth and apoptosis. • GdmCl-induced denaturation process of SphK1 was studied using two different spectroscopic probes. • SphK1 follows the two-state unfolding mechanism i.e., Native (N) ⇌ Denatured (D) state. • Protein aggregation was observed at low concentrations of GdmCl ([GdmCl] ≤ 1.5 M). [ABSTRACT FROM AUTHOR]

Mechanisms of protein aggregation are of immense interest in therapeutic biology and neurodegenerative medicine. Biochemical processes within the living cell occur in a highly crowded environment. The phenomenon of macromolecular crowding affects the diffusional and conformational dynamics of proteins and modulates their folding. Macromolecular crowding is reported to cause protein aggregation in some cases, so it is a cause of concern as it leads to a plethora of neurodegenerative disorders and systemic amyloidosis. To divulge the mechanism of aggregation, it is imperative to study aggregation in well-characterized model proteins in the presence of macromolecular crowder. One such protein is ribonuclease A (RNase A), which deciphers neurotoxic function in humans; therefore we decided to explore the amyloid fibrillogenesis of this thermodynamically stable protein. To elucidate the impact of crowder, dextran-70 and its monomer glucose on the aggregation profile of RNase-A various techniques such as Absorbance, Fluorescence, Fourier Transforms Infrared, Dynamic Light Scattering and circular Dichroism spectroscopies along with imaging techniques like Atomic Force Microscopy and Transmission Electron Microscopy were employed. Thermal aggregation and fibrillation were further promoted by dextran-70 while glucose counteracted the effect of the crowding agent in a concentration-dependent manner. This study shows that glucose provides stability to the protein and prevents fibrillation. Intending to combat aggregation, which is the hallmark of numerous late-onset neurological disorders and systemic amyloidosis, this investigation unveils that naturally occurring osmolytes or other co-solutes can be further exploited in novel drug design strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Urea- and guanidinium chloride (GdmCl)-induced denatured states of horse cytochrome- c (cyt- c) are structurally identical. It is then expected that estimates of ∆ G 0 N→U (Gibbs free energy change in the absence of denaturants) from GdmCl- and urea-induced denaturation curves should be identical, if denaturation induced by them follows a two-state mechanism. That denaturation of cyt- c by urea or GdmCl follows a two-state mechanism is reported by some in vitro studies while other in vitro studies reported contradictory observations. Molecular dynamic (MD) simulation is a technique that could reveal the mechanism of unfolding/folding of proteins in the absence and presence of chemical denaturants at the amino acid residue level. We therefore performed multiple unconstrained MD simulations of cyt- c (PDB ID: 1HRC) in water and aqueous mixtures of GdmCl and urea for the period of 0-500 ns at 300, 400 and 450 K, which showed that denaturation of cyt- c by urea and GdmCl is a two-state and three-state process, respectively. To corroborate these findings, we measured urea- and GdmCl-induced denaturation curves of different optical properties (circular dichroism at 222, 405 and 416 nm and absorbance at 405 nm) and analyzed them for ∆ G 0 N→U. These studies supported conclusions reached from MD simulation studies. Highlights • Cyt- c unfolding by urea and GdmCl was monitored by MD simulations. • Free energy landscapes show that cyt- c unfolding by urea is two-state process. • Free energy landscapes show that cyt- c unfolding by GdmCl is three-state process. • Cyt- c denaturations by urea and GdmCl were monitored by different optical probes. • Results of analysis of denaturation curves corroborate with MD simulation findings. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, overexpressed in cancer cells under hypoxia condition. In cancerous cells, CAIX plays an important role to combat the deleterious effects of a high rate of glycolytic metabolism. In order to favor tumor survival, CAIX maintains intracellular pH neutral or slightly alkaline and extracellular acidic pH. The equilibrium unfolding and conformational stability of CAIX were measured in the presence of increasing urea concentrations to understand it’s structural features under stressed conditions. Two different spectroscopic techniques were used to follow urea-induced denaturation and observed that urea induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of both optical properties suggesting that denaturation of CAIX is a two-state process, i.e., native state ↔ denatured state. Each denaturation curve was analyzed to estimate thermodynamic parameters, Δ G D 0 ,value of Gibbs free energy change (Δ G D ) associated with the urea-induced denaturation, C m (midpoint of denaturation) and m (= δΔ G D /δ[urea]). We further performed molecular dynamics simulation of CAIX for 50 ns to see the dynamics of protein structure in the presence of different urea concentrations. An excellent agreement was observed between in silico and in vitro studies. [ABSTRACT FROM AUTHOR]

• PEG 20 kDa perturbs both secondary as well as tertiary structure of holo a-LA, • PEG 20 kDa induces formation of molten globule in holo a-LA. • ITC measurement revealed a significant binding between holo α-LA and PEG 20 kDa. • This study provided rational to the cause of the deaths occurred in Gambia epidemic. • High dose of glycols in eye drop and cough syrup must be monitored. Macromolecular crowding is driven by hard-core repulsions and soft interactions, may have both the stabilizing and destabilizing effects on protein folding and stability, which has gradually gained attention in recent years. Here, we investigated the impact of polyethylene glycol (PEG), a polymer of ethylene glycol and a constituent of many drugs and eye drops on the structure and stability of holo alpha-lactalbumin (α -LA). Results showed that there was a substantial alteration in the secondary structure of holo α -LA. Further, it was found that PEG disrupted the tertiary structure. In holo α -LA, PEG has been found to create a molten globule state, where the intermediate state contains hydrophobic patches and has a greater hydrodynamic volume than the native protein. Additionally, at the physiological pH, isothermal titration calorimetry revealed a significant binding between holo α -LA and PEG 20,000 Da. We observed that while researching macromolecular crowding, it is extremely important to consider protein binding with crowder and other soft interactions carefully. In this scenario, destabilizing protein-crowder interactions dominated the stabilizing exclusion volume effect. [ABSTRACT FROM AUTHOR]

The macromolecular crowding progressively has been gaining prominence in recent years as it acts as a sword with double-edge on protein stability and folding, i.e., showing assorted results of having both stabilizing and destabilizing effects. We studied the effects of different concentrations of polyethylene glycol (PEG-10) on structure and stability of myoglobin. The tertiary structure of myoglobin was found to be perturbed in the presence of polyethylene glycol, however there was insignificant change in the secondary structure. It was observed that polyethylene glycol induces molten globule state in myoglobin, where the intermediate state holds hydrophobic patches and larger hydrodynamic volume as compared to the native protein. In addition, isothermal titration calorimetry (ITC) showed strong binding between myoglobin and polyethylene glycol, at the physiological pH. We hypothesize that polyethylene glycol induces molten globule conformation in myoglobin by interacting with heme group of myoglobin. We caution that the binding of protein with crowder and other soft interactions need to be gravely well thought-out when studying macromolecular crowding. In our case, destabilizing protein-crowder interactions could compete and overcome the stabilizing exclusion volume effect. [ABSTRACT FROM AUTHOR]

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29 kDa, β-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e. , N (native) ↔ X I ↔ X II ↔ D (denatured) with stable intermediates X I and X II populated around 2 and 4 M urea, respectively. The far-UV CD was used to characterize the intermediate states (X I and X II ) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced X I state has characteristics of molten globule state while X II state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress. [ABSTRACT FROM AUTHOR]

The proper functioning of any protein depends on its three dimensional conformation which is achieved by the accurate folding mechanism. Keeping away from the exposed stress conditions leads to cooperative unfolding and sometimes partial folding, forming the structures like protofibrils, fibrils, aggregates, oligomers, etc. leading to several neurodegenerative diseases like Parkinson's disease, Alzheimer's, Cystic fibrosis, Huntington, Marfan syndrome, and also cancers in some cases, too. Hydration of proteins is necessary, which may be achieved by the presence of organic solutes called osmolytes within the cell. Osmolytes belong to different classes in different organisms and play their role by preferential exclusion of osmolytes and preferential hydration of water molecules and achieves the osmotic balance in the cell otherwise it may cause problems like cellular infection, cell shrinkage leading to apoptosis and cell swelling which is also the major injury to the cell. Osmolyte interacts with protein, nucleic acids, intrinsically disordered proteins by non-covalent forces. Stabilizing osmolytes increases the Gibbs free energy of the unfolded protein and decreases that of folded protein and vice versa with denaturants (urea and guanidinium hydrochloride). The efficacy of each osmolyte with the protein is determined by the calculation of m value which reflects its efficiency with protein. Hence osmolytes can be therapeutically considered and used in drugs. • Diseases occurring due to protein misfolding may be counteracted by natural compounds in order to prevent lethal effects. • Osmolytes like amino acids, polyols and methylamines plays significant role in the mechanism of protein folding. • Functional conformation of protein is achieved through positive interaction of protein and osmolyte. [ABSTRACT FROM AUTHOR]

[Display omitted] • Our study reveals, there is a non-specific binding of fluorescent dyes (ThT & ANS) with crowding agents and their monomers. • Fluorescence measurement studies revealed that there exists strong interaction between the dyes and additives. • ITC and molecular docking experiments also confirmed strong interactions between fluorescent dyes and crowding agents. • The formation of insoluble aggregates of different shapes and sizes was observed under Transmission Electron Microscope. • The degrees of interaction of both dyes were greater with Ficoll 70 than Dextran 70 and their respective monomers. Protein aggregation is a major side reaction that may be observed during protein folding and may prevent proteins from performing their intended functions. It is a hallmark of various human diseases, specifically neurodegenerative diseases. Fluorescence spectroscopy is one such technique in which extrinsic fluorescent dyes are used to monitor protein aggregation in vitro. These dyes are known to get bound to the aggregated portion of proteins, which thereby leads to an enhancement of fluorescence intensity. In this study, we have uniquely investigated with a battery of evidence to show that extrinsic fluorescent dyes bind to macromolecular crowding agents and their monomers even in absence of protein. The additives used in this study are dextran-70 and ficoll-70 and their monomers, glucose and sucrose, respectively. Here, with the help of various biophysical techniques such as extrinsic fluorescent dye-binding assays, molecular docking, ITC and TEM, we have explored the interactions that exist between these extrinsic fluorescent dyes and the macromolecular crowding agents along with their monomers. This study inferred that there is a high possibility of interaction between dyes and additives, as these dyes showed strong binding with above-mentioned crowding agents and their monomers. [ABSTRACT FROM AUTHOR]

The refolding of urea denatured horse heart cytochrome c (h-cyt- c ) under the influence of ester based cationic gemini surfactants [ethane-1, 2-diyl bis( N , N- dimethyl- N- alkylammoniumacetoxy) dichlorides] 16-E2-16, 14-E2-14 and 12-E2-12 ( n- E2- n ) was performed by using UV–visible, fluorescence and circular dichroism (CD) spectroscopic techniques. We found that n- E2- n geminis promote the formation of molten globule (MG) like state upon addition into the urea denatured h-cyt- c . The comparative study of refolding of denatured h-cyt- c with n- E2- n , cationic gemini surfactant show stabilization of MG-like state influenced by hydrophobic interactions. The formation of MG-like state from the unfolded protein confirms the presence of some regular structures induced by n- E2- n gemini surfactants. Thermodynamic parameters for refolding of h-cyt- c by n- E2- n were also measured and the m -values of all the refolded states of h-cyt- c by n- E2- n show marked difference. The higher m -values correspond to the larger hydrophobic chain length indicates that refolding ability of the n- E2- n depends on the alkyl chain length. The result is related to the stronger hydrophobic forces due to the presence of two head groups and two hydrophobic hydrocarbon tails. This study showed that these cationic gemini surfactants were efficiently utilized in the protein refolding studies. [ABSTRACT FROM AUTHOR]

The study of protein misfolding and aggregation saw resurgence in the last decade. Aggregation is the main cause of several human neurodegenerative diseases which makes this field as the leading edge in the science today. Protein aggregation is a highly complex process resulting in formation of a variety of aggregates with different structures and morphologies. Many of them are highly cytotoxic. In-depth knowledge about structure, mechanism of formation, and physiological effects of aggregates will shed new light on the aggregation-mediated cell toxicity, and helps in deciphering new target for drug design and development. This review summarizes the existing information on the molecular mechanism of protein misfolding and aggregation involved in neurodegeneration stressing on the possible therapeutic intervention in neurodegenerative diseases. As our knowledge about the relation between the protein misfolding and disease pathogenesis will be enhanced, more specific and promising treatment opportunities will come into existence. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25 °C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25 M to 3.40 M of the denaturant. However, CAVA gets completely denatured at 4.0 M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40 ns were carried out and a well-defined correlation was established for both in vitro and in silico studies. [ABSTRACT FROM AUTHOR]

Hereditary hemochromatosis factor E (HFE) is a type 1 transmembrane protein, and acts as a negative regulator of iron-uptake. The equilibrium unfolding and conformational stability of the HFE protein was examined in the presence of urea. The folding and unfolding transitions were monitored with the help of circular dichroism (CD), intrinsic fluorescence and absorption spectroscopy. Analysis of transition curves revealed that the folding of HFE is not a two-state process. However, it involved stable intermediates. Transition curves (plot of fluorescence (F 346 ) and CD signal at 222 nm (θ 222 ) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (C m ) values at 2.88 M and 4.95 M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96 M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea ( Δ G D 0 ) associated with the equilibrium of denaturation exist between native state ↔ denatured state. The intermediate state was further characterized by hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-binding). For seeing the effect of urea on the structure and dynamics of HFE, molecular dynamics simulation for 60 ns was also performed. A clear correspondence was established between the in vitro and in silico studies. [ABSTRACT FROM AUTHOR]

Sheep serum albumin (SSA) is a 583 amino acid residues long multidomain monomeric protein which is rich in cysteine and low in tryptophan content. The serum albumins (from human, bovine and sheep) play a vital role among all proteins investigated until now, as they are the most copious circulatory proteins. We have purified SSA from sheep kidneys by a simple and efficient two-step purification procedure. Further, we have studied urea-induced denaturation of SSA by monitoring changes in the difference absorption coefficient at 287 nm (Δ ε 287 ), intrinsic fluorescence emission intensity at 347 nm ( F 347 ) and mean residue ellipticity at 222 nm ([ θ ] 222 ) at pH 7.4 and 25 °C. The coincidence of denaturation curves of these optical properties suggests that urea-induced denaturation is a bi-phasic process (native (N) state ↔ intermediate (X) state ↔ denatured (D) state) with a stable intermediate populated around 4.2–4.7 M urea. The intermediate (X) state was further characterized by the far-UV and near-UV CD, dynamic light scattering (DLS) and fluorescence using 1-anilinonaphthalene-8-sulfonic acid (ANS) binding method. All denaturation curves were analyzed for Gibbs free energy changes associated with the equilibria, N state ↔ X state and X state ↔ D state in the absence of urea. [ABSTRACT FROM AUTHOR]

Cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue exists in two natural forms named as full length (FL-αC-PE) and truncated (Tr-αC-PE). FL-αC-PE and Tr-αC-PE are produced when cyanobacterium is grown in the optimal medium and nutrient deficient medium, respectively. Despite of N-terminal deletion, both proteins show similar spectroscopic properties. In this study, different optical properties of these two natural variants of C-PE were measured in the pH range 1.0–12.0 (1.0 ≤ pH ≤ 12.0). It was observed that: (i) their absorption, fluorescence and CD spectra remain unchanged within the range adjacent to neutral pH, 5.5–8.75, (ii) at pH values higher than 8.75 and lower than 5.5 their absorption, fluorescence and CD spectral signatures are changed significantly, and (iii) emission spectra of the covalently linked tetrapyrrole chromophores and Trp residue are perturbed at extreme pH values in the range 8.75 < pH < 5.5. Refolding experiments further suggest that pH-induced denaturation of both forms of C-PE is reversible in the pH range 2.5–11.0, but irreversible beyond this range on both sides of pH extremes. The pH-induced denaturation of both the full length and truncated αC-PEs follows a two-state mechanism. [ABSTRACT FROM AUTHOR]

Pioneer works to understand protein-folding problem have been done in dilute solutions. However, the cellular environment is crowded with macromolecules where excluded volume affects protein stability and function. Here, we assess thermal denaturation curves of RNase A in the presence of various concentrations of dextran 70 at different pH values. Values of thermodynamic parameters such as T (midpoint of denaturation), ∆ H (enthalpy change at T) and ∆ C (constant pressure heat capacity change) were determined under a given solvent condition. Using values of T, ∆ H and ∆ C in Gibbs-Helmholtz equation, $$\Delta G_{\text{D}}^{ \circ }$$ (Gibbs energy change at 25 °C) was determined. It has been observed that dextran 70 stabilizes RNase A in terms of T and $$\Delta G_{\text{D}}^{ \circ }$$ . The stabilization of the protein is more at lower pH where the effect of exclusion is more. We had also investigated the secondary and tertiary structure of RNase A in the presence of dextran 70 and found that dextran 70 induces secondary structure, which is more prominent at physiological pH. However, there is no effect of crowding agent on tertiary structure of the protein. Our study demonstrates that crowding enhances thermal stability as well as the secondary structure of the protein. [ABSTRACT FROM AUTHOR]

The oligonucleotide binding fold (OB-fold) is a small structural motif present in many proteins. It is originally named for its oligonucleotide or oligosaccharide binding properties. These proteins have been identified as essential for replication, recombination and repair of DNA. We have successfully purified a protein contains OB-fold from the stem of Tinospora cordifolia , a medicinal plants of north India. Stems were crushed and centrifuged, and fraction obtained at 60% ammonium sulphate was extensively dialyzed and applied to the weak anion exchange chromatography on Hi-Trap DEAE-FF in 50 mM Tris–HCl buffer at pH 8.0. Eluted fractions were concentrated and applied to gel filtration column to get pure protein. We observed a single band of 20-kDa on SDS-PAGE. Finally, the protein was identified as OB-fold by MALDI-TOF. The purified OB-fold protein was characterized for its secondary structural elements using circular dichroism (CD) in the far-UV region. Generally the OB-fold has a characteristic feature as five-stranded beta-sheet coiled to form a closed beta- barrel. To estimate its chemical stability, guanidinium chloride-induced denaturation curve was followed by observing changes in the far-UV CD as a function of the denaturant concentration. Analysis of this denaturation curve gave values of 8.90 ± 0.25 kcal mol −1 and 3.78 ± 0.18 M for Δ G D ° (Gibbs free energy change at 25 °C) and C m (midpoint of denaturation), respectively. To determine heat stability parameters of OB-fold protein, differential scanning calorimetry was performed. Calorimetric values of Δ G D °, T m (midpoint of denaturation), Δ H m (enthalpy change at T m ), and Δ C p (constant-pressure heat capacity change) are 9.05 ± 0.27 kcal mol −1 , 85.2 ± 0,3 °C, 105 ± 4 kcal mol −1 and 1.6 ± 0.08 kcal mol −1 K −1 . This is the first report on the isolation, purification and characterization of OB-fold protein from a medicinal plant T. cordifolia. [ABSTRACT FROM AUTHOR]

[Display omitted] • Formation of pre molten globule (PMG) in cyt c in presence of dextran (polymer) due to soft interactions. • Glucose (monomer) induces molten globule (MG) in cyt c due to volume exclusion phenomenon. • Presence of PMG and MG in a same protein under different environmental conditions. • Crowders induce different conformational intermediates in similar environment as evident by interaction studies (ITC & MD). Protein attains its biological functional state in a crowded environment, whereas traditional in vitro experiments are carried out in dilute solutions. In this investigation, synthetic crowders (dextran and glucose) are exploited to investigate the effect of crowding on the conformation of cytochrome c. Protein folding may occur through many non-native intermediate states. Here, the effect of crowder (dextran) and its monomer (glucose) on the structural conformation and stability of cytochrome c (cyt c) has been studied in an acidic environment using various biophysical techniques such as UV–Vis absorbance, circular dichroism, and fluorescence spectroscopy (Trp and ANS fluorescence), dynamic light scattering and interaction experiments using ITC and molecular docking. These techniques show that the cyt c forms a molten globule in the presence of glucose while the pre-molten globule in the presence of the dextran 70. These results suggest that dextran 70 stabilizes the protein due to macromolecular crowding via soft interactions, while glucose due to co-solute engineering exploiting volume exclusion phenomenon as confirmed by ITC experiments. These investigations inferred that crowders protect protein in harsh conditions (acidic pH), which may arise due to various diseased conditions such as acidosis-induced insulin resistance, cardiovascular risks, etc. The current work also has an industrial application to increase the shelf life of therapeutic proteins. [ABSTRACT FROM AUTHOR]

To understand the role of five extra N-terminal residues, we prepared wild type (WT) yeast iso-1-cytochrome c (y-cyt- c ) and its deletants by subsequently deleting these residues. Denaturation of all these proteins induced by LiCl was followed by observing changes in molar absorption coefficient at 405 nm (Δ ɛ 405 ), the mean residue ellipticity at 222 nm ([ θ ] 222 ), and the difference mean residue ellipticity at 409 nm (Δ[ θ ] 409 ) near physiological pH and temperature (pH 6.0 and 25 °C). It was observed that in each case LiCl induces biphasic transition, N (native) state ↔ X (intermediate) state ↔ D (denatured) state. The intermediate (X) was characterized by the far-UV, near-UV and Soret circular dichroism, ANS (8-anilino-1-naphthalenesulfonic acid) binding and dynamic light scattering measurements. These measurements led us to conclude that X state of each protein has structural characteristics of PMG (pre-molten globule) state. Thermodynamic stability of all proteins was also determined. It was observed that the N-terminal extension stabilizes the native WT protein but it has no effect on the stability of PMG state. Another state was observed for each protein, in the presence of 0.33 M Na 2 SO 4 at pH 2.1, which when characterized showed all structural characteristics of MG (molten globule) state. [ABSTRACT FROM AUTHOR]

Abstract: In practice, the observation of non-coincidence of normalized sigmoidal transition curves measured by two different structural properties constitutes a proof of existence of thermodynamically stable intermediate(s) on the folding ↔ unfolding pathway of a protein. Here we give first experimental evidence that this non-coincidence is also observed for a two-state protein denaturation. Proof of this evidence comes from our studies of denaturation of goat cytochrome-c (g-cyt-c) at pH 6.0. These studies involve differential scanning calorimetry (DSC) measurements in the absence of urea and measurements of urea-induced denaturation curves monitored by observing changes in absorbance at 405, 530, and 695nm and circular dichroism (CD) at 222, 405, and 416nm. DSC measurements showed that denaturation of the protein is a two-state process, for calorimetric and van’t Hoff enthalpy changes are, within experimental errors, identical. Normalization of urea-induced denaturation curves monitored by optical properties leads to noncoincident sigmoidal curves. Heat-induced transition of g-cyt-c in the presence of different urea concentrations was monitored by CD at 222nm and absorption at 405nm. It was observed that these two different structural probes gave not only identical values of T m (transition temperature), Δ H m (change in enthalpy at T m) and Δ C p (constant-pressure heat capacity change), but these thermodynamic parameters in the absence of urea are also in agreement with those obtained from DSC measurements. [Copyright &y& Elsevier]

[Display omitted] • Polyethylene glycol (PEG 4 kDa) induces two different intermediate states in myoglobin (Mb). • The intermediate states were characterized using spectroscopic techniques and MD simulations. • This study showed a protein does not follow a singular and specific pathway during folding. • This study foresees the energy landscape theory. • This study shows significance of crowding concentration in cellular organism. Practically proteins perform their functions exposed to various molecules (macro/micro) of different shapes, sizes, and high concentrations. In such conditions, proteins fold through various intermediate states to execute their type of functions. There are various in vitro studies that showed that proteins yield intermediate states (either pre-molten globule, PMG or molten globules, MG) in changing environments (pH, temperature, and co-solute). Here, we investigate that myoglobin (Mb) yields two intermediate states in the presence of polyethylene glycol, PEG (4 kDa molecular weight) at two different concentrations. These intermediates were characterized by various spectroscopic techniques, further; we demonstrated that these changes in the structure of the protein were due to soft interactions which were confirmed by isothermal titration calorimetric and computational studies. Besides, in silico (molecular dynamic simulations) studies were exploited to know the atomic-level details of the protein which are useful to comprehend the functional characteristics of the bio-molecule with structural change and to study atomic motions and inter-atomic interactions in the bio-molecular systems. This is the first time that two intermediates (MG and PMG state) in the protein (Mb) at two different concentrations in the presence of solo size of PEG are yielded. This study shows folding of a protein does not follow a singular and specific pathway but occurs through routes down a folding funnel more like rain flowing down a funnel, hence foresees the energy landscape theory. Moreover, the study provides the significance of crowding concentrations in the cellular organism. [ABSTRACT FROM AUTHOR]