Pre-molten Globule State Research Articles

The physical basis of fabrication of amyloid-based hydrogels by lysozyme.

The fabrication of amyloid-based hydrogels has attracted remarkable attention within the field of materials science and technology. These materials have a multitude of potential applications in the biomaterials field such as developing scaffolds for tissue engineering, drug delivery and hygiene products. Despite the potential new applications of these materials, the physical nature of their assembly is not well understood. In this study, we have investigated how the conformation of the amyloid precursor state (I) is formed and correlated with the assembly of amyloid-based hydrogels. A transparent hydrogel was fabricated at pH 7.4 by cooling of the temperature-induced unfolded state of hen egg white lysozyme (HEWL). The completely unfolded state (U) at the gelation concentration of HEWL was obtained around 90 °C in the presence of tris(2-carboxyethyl)phosphine (TCEP), with a TCEP/HEWL molar ratio of 4 : 1. The characterization of the hydrogel showed that it was composed of an amyloid fibril-like material. The physical nature of its assembly was examined in detail and it was found that the hydrogel formation reaction was a three-step, four-states process (U → I → F → H). We concluded that the properties of the pre-molten globule state (I) of the protein correlated only with the fibrillation process, whereas the assembly of the fibrils into an hydrogel was found to be almost independent of the I state. Thus, the study presented here provides a complete biophysical insight into the pathway of lysozyme hydrogel assembly.

Conformational diversity in the intrinsically disordered HIV-1 Tat protein induced by zinc and pH

Human immunodeficiency virus type-1 (HIV-1) transactivator of transcription (Tat) is an intrinsically disordered protein that exerts multiple functions, including activation of HIV-1 replication and induction of T-cell apoptosis and cytokine secretion via zinc binding and cellular uptake by endocytosis. However, the effects of zinc and endosomal low pH on the structure of isolated Tat protein are poorly understood. Here, we purified a monomeric zinc-bound Tat and studied its structure and acid denaturation by circular dichroism, NMR, and small-angle X-ray scattering. We found that at pH 7, the zinc-bound Tat was in a pre-molten globule state; it exhibited largely disordered conformations with residual helices and was slightly more compact than the fully unfolded states that were observed at pH 4 or in the zinc-free form. Moreover, acid-induced unfolding transitions in secondary structure and molecular size occurred at different pH ranges, indicating the presence of an expanded and helical intermediate at pH ∼6. Taken together, the extent of structural disorder in the intrinsically disordered Tat protein is highly sensitive to zinc and pH, suggesting that zinc binding and pH affect Tat structures and thereby control the versatile functions of Tat.

First evidence of formation of pre-molten globule state in myoglobin: A macromolecular crowding approach towards protein folding in vivo

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.

Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29kDa, 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) ↔ XI ↔ XII ↔ D (denatured) with stable intermediates XI and XII populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) 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 XI state has characteristics of molten globule state while XII 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.

Rifampicin Induced Aggregation of Ovalbumin: Malicious Behaviour of Antibiotics.

Molecular modeling deciphered the site of interaction of rifampicin in the structure of ovalbumin at a site which is surrounded by residues Glu-214, Asp-98, Pro-85, Asp-91 and Asp-47. Isothermal calorimetric analysis determined the thermodynamic parameters i.e. ΔH and ΔS which came out be -8.086 cal/mol and -131 cal/mol/deg. respectively. Ovalbumin is a secretory protein of hen oviduct, present in the human blood serum and interacts with the drug rifampicin in vivo, when administered. Simulating these conditions in vitro revealed that rifampicin induced the aggregated state at 6 µM concentration which was featured by a decrease in the ANS fluorescence intensity relative to the native state while as the pre-molten and molten globule state were obtained at 3 µM and 5 µM concentration of rifampicin respectively displaying a hike in the ANS fluorescence intensity. Far-UV CD analysis suggested β-sheet rich structure with negative ellipticity peak at 217 nm for native ovalbumin incubated with 6 µM rifampicin. Increase in absorbance at 450 nm, red shift of 50 nm in the congo red binding assay and a hike of 10 fold in the ThT fluorescence intensity compared to the native state further confirmed aggregate formation. Moreover, TEM images displayed aggregates to be spherical morphologically. Aggregates formed at 6 µM rifampicin concentration were found to be cytotoxic as there was a reduction of cell viability to 28%. Thus, protein-drug interaction primarily facilitates a structural alteration in the native structure of proteins leading to their aggregation which were proved to be cytotoxic in nature.

Characterization of pre-molten globule state of yeast iso-1-cytochrome c and its deletants at pH 6.0 and 25 °C

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 405nm (Δɛ405), the mean residue ellipticity at 222nm ([θ]222), and the difference mean residue ellipticity at 409nm (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 0.33M Na2SO4 at pH 2.1, which when characterized showed all structural characteristics of MG (molten globule) state.

An insight into the biophysical characterization of different states of cefotaxime hydrolyzing β-lactamase 15 (CTX-M-15)

Cefotaxime hydrolyzing β-lactamase-15 (CTX-M-15) is encoded by blaCTX-M-15 gene present on plasmid of various Gram-negative bacteria, such as E. coli, E. cloacae, K. pneumoniae, etc. The widespread dissemination of CTX-M-15 harboring bacteria in hospital as well as community settings is a universal threat as they are resistant to various clinically significant antibiotics. In order to gain an insight into the folding mechanism of CTX-M-15, we carried out pH-induced denaturation study by monitoring Trp fluorescence, far-UV circular dichroism (CD), and ANS fluorescence. We found that the pH-induced denaturation of CTX-M-15 was a three-step process with the accumulation of two stable folding intermediates (XI at pH 2.5 and XII at pH 1.5) in the folding pathway. The intermediates were further characterized by far-UV and near-UV CD analysis, Trp fluorescence, ANS fluorescence, three-dimensional fluorescence, acrylamide quenching, dynamic light scattering, and thermal denaturation studies. We found that XI state lacked tertiary structure but retained most of the secondary structure, its Trp residues were partially exposed to the solvent and its hydrophobic patches were highly accessible to ANS. On the other hand, a complete disruption of tertiary structure along with more than 50% loss in secondary structure was observed in XII state. We conclude that the XI state of CTX-M-15 at pH 2.5 had all the characteristics of a molten globule (MG) state, while its XII state at pH 1.5 was more similar to pre-molten globule (PMG) state. ANS fluorescence also showed that the binding of ANS in XII state was lower than that in the XI state. We propose that the accumulation of MG- and PMG-states was due to separation (at pH 2.5) and then unfolding (at pH 1.5) of the αβα-fold of CTX-M-15, respectively.

Induction of amyloidogenicity in wild type HEWL by a dialdehyde: Analysis involving multi dimensional approach

Physiological conditions corresponding to oxidative stress deplete the level of enzyme glyoxalase, facilitating a hike in the serum concentration of glyoxal. Simulating an elevated in vivo level of glyoxal, we tested (50%, v/v) concentration of glyoxal to interact with HEWL. Initially, docking study revealed that glyoxal binds in the hydrophobic core of the enzyme. The interaction between the dialdehyde (glyoxal) and the enzyme (HEWL) followed a three step transition involving pre-molten and molten globule states formed on days 7 and 15 of incubation respectively, which were characterised by an increase in the ANS fluorescence intensity compared to the native state. These molten globule states upon further incubation on day 20 resulted in the formation of aggregates which were characterised by an increase in ThT fluorescence intensity, red shift in Congo red absorbance, negative ellipticity peak at 217nm in the far-UV CD and the loss of signals at 284, 290 and 294nm in the near-UV CD spectra. Finally, TEM confirmed the authenticity of lysozyme fibril formation by displaying rod like fibrillar structure.

Conformational Analysis of Isolated Domains of Helicobacter pylori CagA

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation - a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

Dynamics and dimension of an amyloidogenic disordered state of human β2-microglobulin

Human β2-microglobulin (β2m) aggregation is implicated in dialysis-related amyloidosis. Previously, it has been shown that β2m adopts an ensemble of partially unfolded states at low pH. Here we provide detailed structural and dynamical insights into the acid unfolded and yet compact state of β2m at pH2.5 using a host of fluorescence spectroscopic tools. These tools allowed us to investigate protein conformational dynamics at low micromolar protein concentrations in an amyloid-forming condition. Our equilibrium fluorescence data in combination with circular dichroism data provide support in favor of progressive structural dissolution of β2m with lowering pH. The acid unfolded intermediate at pH2.5 has high 8-anilinonaphthalene, 1-sulfonic acid (ANS)-binding affinity and is devoid of significant secondary structural elements. Using fluorescence lifetime measurements, we have been able to monitor the conformational transition during the pH transition from the native to the compact disordered state. Additionally, using time-resolved fluorescence anisotropy measurements, we have been able to distinguish this compact disordered state from the canonical denatured state of the protein by identifying unique dynamic signatures pertaining to the segmental chain mobility. Taken together, our results demonstrate that β2m at pH2.5 adopts a compact noncanonical unfolded state resembling a collapsed premolten globule state. Additionally, our stopped-flow fluorescence kinetics results provide mechanistic insights into the formation of a compact disordered state from the native form.

SDS Induced Refolding of Pre-molten Globule State of Cryptolepain: Differences in Chemical and Temperature-Induced Equilibrium Unfolding of the Protein in SDS-Induced State

Interaction of sodium dodecyl sulfate (SDS) with a globular protein cryptolepain to induce stabilization of the pre-molten globule state of the protein under acidic condition was studied. At pH 2.0, cryptolepain is in partially unfolded state with native like secondary structure, exposed hydrophobic surfaces without any rigid tertiary structure. Unfolding of cryptolepain in the pre-molten globule state (state at pH 2.0) is co-operative suggesting that the protein unfolds as a single entity. Addition of SDS below its critical micelle concentration of 2.0 mM at acidic pH drives the protein to a non-native state similar to molten globule state seen with other proteins. Temperature induced unfolding of such SDS-induced state of the cryptolepain is biphasic suggesting that the molecular structure of the protein is constituted of two structural entities which are stabilized differentially in the presence of SDS and unfold sequentially. Whereas the chemical induced unfolding of SDS-induced state of cryptolepain is co-operative with higher transition midpoints indicating that the protein is stabilized as a whole. Unfolding of SDS-induced state of cryptolepain is very similar to the unfolding of the cryptolepain at very low pH (0.5), where the protein exists in molten globule state. Thus, SDS interactions result in the refolding of the acid unfolded state of the protein and drive the protein into a non-native state similar to the molten-globule state seen at very low pH.

Structural disorder and local order of hNopp140

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography, 1H NMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0–80% TFE suggests a tendency to form local helical structures. Wide-line NMR measurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).

Solution Model of the Intrinsically Disordered Polyglutamine Tract-Binding Protein-1

Polyglutamine tract-binding protein-1 (PQBP-1) is a 265-residue nuclear protein that is involved in transcriptional regulation. In addition to its role in the molecular pathology of the polyglutamine expansion diseases, mutations of the protein are associated with X-linked mental retardation. PQBP-1 binds specifically to glutamine repeat sequences and proline-rich regions, and interacts with RNA polymerase II and the spliceosomal protein U5-15kD. In this work, we obtained a biophysical characterization of this protein by employing complementary structural methods. PQBP-1 is shown to be a moderately compact but largely disordered molecule with an elongated shape, having a Stokes radius of 3.7 nm and a maximum molecular dimension of 13 nm. The protein is monomeric in solution, has residual β-structure, and is in a premolten globule state that is unaffected by natural osmolytes. Using small-angle x-ray scattering data, we were able to generate a low-resolution, three-dimensional model of PQBP-1.

Analysis of the molten globule state of bovine α-lactalbumin by using vibrational circular dichroism

We investigated molten globule state of α-lactalbumin by using vibrational circular dichroism (VCD) and two-dimensional (2D) correlation spectroscopy. To investigate details on the denaturation mechanism of α-lactalbumin, 2D correlation spectroscopy was applied to the pH-dependent VCD spectra of α-lactalbumin aqueous solutions. The denaturation transition point of α-lactalbumin was determined by 2D gradient mapping method. Large spectral changes of VCD spectra were observed at pH 4.75 and 2.75 in 2D gradient map, which are transition points. Based on analysis of 2D VCD correlation spectra, we can suggest the sequence change from pre-molten globule state to molten globule state. Intermolecular β-sheet changed first, and then lower-frequency β-sheet structure changed before α-helices with continued lowering of the pH.

PH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.

Protein folding and the order/disorder paradox

Most proteins encoded by the nuclear genome are synthesized in the cytoplasm and fold into precise 3D structures. During synthesis, the nascent polypeptide begins to fold as it traverses the large subunit of the ribosome and is assisted by molecular chaperones in attaining its precise folded/highly ordered state efficiently and in a biologically relevant timescale. Proteins that are misfolded are culled, re-routed, and marked by mechanisms such as ubiquitinylation for degradation ensuring strict quality control (QC). In addition to the highly ordered "globular" proteins, emerging evidence indicates that a large fraction of the proteome also comprises the so-called "Intrinsically Disordered Proteins" (IDPs). IDPs are proteins that lack rigid 3D structures and instead, exist as dynamic ensembles. The dynamic structures in the IDPs have many similarities with "normal" globular proteins such as the native (ordered), and non-native (molten globule, pre-molten globule, and coil-like) states seen during folding of "normal" globular proteins. However, unlike the case of the nascent globular proteins, IDPs evade being detected as "misfolded" and degraded by the cell's QC system. We refer to this paradox as the order/disorder paradox and postulate that the IDPs capitalize on their intrinsic promiscuity and ability to undergo disorder-to-order transitions upon binding to biological targets (coupled folding and binding) to escape the cell's surveillance machinery. Understanding the mechanism by which the IDPs evade the quality check has wide implications from protein folding to disease biology since the aggregation of misfolded proteins underlies several debilitating illnesses such as many neurodegenerative diseases and cancer.

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Unfolded and partially folded states of proteins play a crucial role in the formation of fibril aggregates in human neurodegenerative diseases. Some disease-associated proteins adopt well-folded structures in their native states while others belong to the class of intrinsically unstructured proteins. The conversion of a soluble form of a protein to the aggregated state is typically related to the transient formation of unfolded or partially folded states for both classes of proteins. In many protein-folding diseases, single-point mutations, often inherited, are linked to specific disease forms but the structural code for the onset of the disease is still unclear. Single-point mutations do not significantly change the structure of the native state. Their impact on the structural and dynamic characteristics of the unfolded or intrinsically unstructured state ensemble remains a key unsolved question in structural biology. One of the approximately 20 amyloid diseases known to date is associated with the deposition of amyloid plaques from single-point mutants (I56T, F57I, I59T, W64R, and D67H) of human lysozyme (hLys) in various organs. Given the remarkable high stability of this protein family, the discovery of protein-folding diseases associated with hLys came as a surprise. Initial studies revealed that the native-state structures of wildtype (wt) and amyloidogenic mutants of hLys are highly similar. Characterization of the thermal unfolding at pH 1.2 using CD spectroscopy, differential scanning calorimetry (DSC), and NMR spectroscopy showed that unfolding proceeds through loss of tertiary structure, resulting in a premolten globule state, which unfolds further as the temperature increases. The b domain and the adjacent helix 3 (residues 90–100) unfold first. The same region is destabilized in the mutants as shown by hydrogen exchange, leading to lower thermal stability. This decrease in stability suggests that the relative stabilities of the folded and unfolded states are important in the process of amyloid formation. In fact, amyloid fibrils are formed fastest when the protein is incubated at very low pH (1.2) and at temperatures near the midpoint of thermal unfolding, thus at a temperature at which folded as well as molten globule and premolten globule states, referred to here as unfolded states, are populated. For a better understanding of the mechanism leading to the formation of amyloid diseases, both folded and unfolded states therefore need to be characterized. In contrast to the folded state of hLys, little is known about the residual structure and dynamics of its nonnative unfolded state. Heteronuclear NMR spectroscopy permits the characterization of not only the structure but also the dynamics of unfolded nonnative protein ensembles at atomic resolution. To highlight the remarkable influence of the mutants on the structure and dynamics in unfolded hLys, we first compare the unfolded states of the hen egg white lysozyme (hewl)and hLys to highlight the intrinsic higher aggregation propensity of hLys relative to that of hewl, and then discuss how two amyloidogenic mutants modulate the

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

Proteins in cells fold via a number of intermediates. These intermediates are quite important as they guide the protein to attain its unique native conformation. To solve the immensely difficult problem of protein folding, it is necessary to characterize intermediates which will unravel the mystery of the steps involved in the proper folding of proteins. Cytochromes-c (cyts-c) have played an important role in studies of the earliest events and intermediates in protein folding. They have always been considered as model proteins for protein folding studies due to their intrinsic properties that can be measured by multiple probes. A large number of different solvent conditions have been employed to obtain equilibrium intermediates of cyts-c. These intermediates show structural heterogeneity which is mainly due to the different solvent conditions used to induce them. In this review we present results of conformational and thermodynamic characterization of equilibrium intermediates (molten globules and pre-molten globules) of the mammalian cyts-c under different solvent conditions.

Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c

It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol(-1), whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol(-1). A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.