How Do You Know Whether You Are Observing a Molten Globule State

Biophys Rev. 2019 Jun; 11(three): 365–375.

A look back at the molten globule country of proteins: thermodynamic aspects

Eva Judy

Department of Chemistry, Indian Institute of Engineering science Mumbai, Powai, Mumbai, 400 076 Bharat

Nand Kishore

Department of Chemistry, Indian Constitute of Engineering science Bombay, Powai, Mumbai, 400 076 India

Received 2019 Apr 1; Accepted 2019 Apr 22.

Abstruse

Involvement in poly peptide folding intermediates lies in their significance to protein folding pathways. The molten globule (MG) state is one such intermediate lying on the kinetic (and sometimes thermodynamic) pathway between native and unfolded states. Development of our qualitative and quantitative understanding of the MG country can provide deeper insight into the folding pathways and hence potentially facilitate solution of the protein folding trouble. An extensive look at literature suggests that nearly studies into protein MG states accept been largely qualitative. Attempts to obtain quantitative insights into MG states have involved awarding of high-sensitivity calorimetry (differential scanning calorimetry and isothermal titration calorimetry). This review addresses the progress made in this direction by discussing the knowledge gained to date, along with the future promise of calorimetry, in providing quantitative information on the structural features of MG states. Particular attention is paid to the question of whether such states share common structural features or not. The divergence in the nature of the transition from the MG state to the unfolded state, in terms of cooperativity, has too been addressed and discussed.

Keywords: Molten globule state, Protein folding, Isothermal titration calorimetry, Differential scanning calorimetry, Thermal transitions, Thermodynamic signatures

The molten globule state

The term molten globule was coined by Ohgushi and Wada in 1983 (Ohgushi and Wada 1983), with due credit to O. B. Ptitsyn and C. Crane-Robinson about discussion on this terminology. The traditional definition of the molten globule (MG) state describes information technology as a compact intermediate in which the third structure of the protein is lost but the secondary structure is intact or even strengthened (Kuwajima 1989). An often-used alternative definition of the MG land is "a land in which specific third structure is disrupted without loss of the secondary structure" (Ptitsyn 1987; Kuwajima 1989). The MG state can therefore be considered similar to the partially folded land, sometimes observed during the unfolding of a protein. In the early on 1990s, interest in the MG state arose jointly due to its presumed similarity with the intermediate land in poly peptide folding, and as a translocation competent country in the transport of a precursor poly peptide beyond biological membranes (Bychkova et al. 1988; Martin et al. 1991; Van der Goot et al. 1991). Information technology was felt that thermodynamic characterization of MG states may provide fundamental information in unraveling the protein folding problem.

Different models take been proposed to explain protein folding in the past, some of which are the framework model, the nucleation model, the improvidence collision model, the hydrophobic plummet model, and the ensemble new view models. The framework model proposed past Ptitsyn (1973) considered the germination of secondary structural elements followed by the formation of farther avant-garde folding intermediate and and then specific packing of the side chains (Udgaonkar and Baldwin, 1988). This model suggested the being of several folding intermediates during the folding process (Ptitsyn and Rashin 1975; Kim and Baldwin 1982; Roder et al. 1988). A few years afterward, Karplus and Weaver (1976) assumed that many unstable quasiparticles found the intact protein. The quasiparticles are likewise known as microdomains which are portions of nascent secondary structures and hydrophobic clusters. In order to achieve stability, the microdomains diffuse, collide, and eventually coalesce. Hydrophobic collapse was predicted as an early on event in the folding procedure (Get 1984; Agashe et al. 1995; Rackovsky and Scheraga 1977; Dill 1985; Arai et al. 2007) later it was recognized that the interior of the native states of proteins unremarkably contains a hydrophobic cadre of nonpolar amino acid residues. On similar lines, collapse around a diffuse nucleus was considered to propose the nucleation condensation model assuming that the limiting step in the folding of a protein is nucleus formation (Fersht 1997). The nucleus formation is followed by a fast propagation of the structure. The primary characteristic of the nucleation condensation model is that it proposes the formation of simultaneous secondary and tertiary interactions (Nölting and Agard 2008).

The new views on protein folding models propose energy landscape and folding funnel models (Bryngelson et al. 1995; Onuchic et al. 1997) based on the dependence of free energy on the coordinates that determine poly peptide conformation. According to this most adopted model, the folding of a protein from the highest energy level disordered land flows down a funnel, passing through intermediates towards global energy minima which corresponds to its native conformation. The zipper model (Dill et al. 1993; Munoz et al. 1997) suggests a zipper-like folding procedure whereas the funnel model emphasizes on parallel pathways of folding (Wolynes et al. 1995; Onuchic et al. 1996). Molten globule is represented as 1 of the intermediate states in the funnel model (Onuchic et al. 1996).

Resemblance of the kinetic intermediates formed during poly peptide folding with the equilibrium MG state for some proteins such every bit apomyoglobin and α-lactalbumin has been discussed earlier (Barrick and Baldwin 1993; Jennings and Wright 1993; Balbach et al. 1995; Forge et al. 1999). The importance of the MG land and like other nonnative states of the protein in their transition to the MG land has been recognized (Bychkova et al. 1988; Penkett et al. 1998; Kelly 1998; Chiti et al. 1999). The role of the MG land of human α-lactalbumin in apoptosis in tumor cells has also been reported (Svensson et al. 1999).

Structural studies of MG states obtained under equilibrium weather for a diversity of proteins raised the full general question of whether these intermediate states are a universal feature of protein behavior (Haynie and Freire 1993). Another term, the "pre-molten globule state" was also coined in the early 1990s (Jeng and Englander 1991). Like the MG country, the pre-molten globule had intact elements of secondary structure, but dissimilar the MG state, it was non considered compact. Articles did appear almost the solvation dynamics (Samaddar et al. 2006) and conformation and thermodynamic stability of the pre-MG country (Khan et al. 2011) along with various models for MG states (Fink et al. 1998), merely the information available from these studies remained qualitative in nature. With the passage of time, the interest in the characterization of the pre-MG land declined significantly and currently there are hardly any reports which address such pre-MG states.

Recently, Takahashi et al. (2018) take hypothesized structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and long-range altitude distribution. These authors observed peak broadening in the fluorescence resonance energy transfer (FRET) efficiency plot for the unfolded proteins and suggested the significance of local heterogeneous clusters in the unfolded states of proteins. Obtaining further insights into such clusters could be important in understanding the machinery of protein folding. FRET methods have also been practical in studies of intrinsically matted proteins in statistical terms (Haas 2012). Information technology was suggested that the decision of intramolecular distance distributions by ensemble and single-molecule FRET experiments enables exploration of partially folded states in the refolding of protein molecules (Haas 2005).

Sasahara et al. (2000) observed a partially unfolded equilibrium state of hen egg white lysozyme based on circular dichroism spectroscopic measurements. They further observed that the transition from intermediate to unfolded land is associated with low cooperativity and small enthalpy and entropy changes. These authors did non explicitly use the term MG country, merely suggested that the observed intermediate has characteristics of kinetic intermediates observed in the refolding pathway of hen lysozyme.

Conception and progress in understanding MG states

Even though the poly peptide folding problem was addressed as early every bit the 1960s (Perutz et al. 1960; Anfinsen et al. 1961; Haber and Anfinsen 1962; Dill and MacCallum 2012), the intermediate states, especially the MG state, only began receiving attending from the early on 1980s; however, the number of enquiry articles published per year was less than 10 upwards to the year 1990. Figure 1 suggests that the scattered data nearly such a state in the 1980s did non produce meaning interest in the scientific community. Nevertheless, a sharp increase in enquiry on the MG country from 1990 to 1996 indicated a growing understanding of the importance of the MG conformation. From the twelvemonth 1990 onwards, up to 2000, the number of inquiry manufactures published on MG states increased significantly (see Fig.1).

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Number of publications addressing the molten globule country in proteins from the early 1980s up to 2018 representing the rise and decline in scientific output on the cognition of intermediate states (source: Web of Science)

Equally judged by Fig. 1, the corporeality of scientific literature on the MG states started declining after the year 2000. This could mean that either a deeper understanding of the MG country is not important or it is becoming extremely difficult to obtain new data on this country over and above the existing knowledge. In view of the tough task of obtaining more insights and relevance of the MG state, rigorous efforts need to be continued to understand the role of the MG state not but in the poly peptide folding merely also in its connection to aggregation and fibrillization of the proteins (Ptitsyn 1973; Hammarströ et al. 1999; Chiti et al. 1999; Povarova et al. 2010; Skora et al. 2010).

Characterization of the MG state of the protein has mostly been done qualitatively by circular dichroism spectroscopy, a technique capable of providing direct prove on the extent of loss of tertiary structure and retaining/strengthening of the secondary structure (Vassilenko and Uversky 2002). Measurement of the intrinsic fluorescence on proteins has likewise provided much supporting information on the MG land (Swaminathan et al. 1994). For dye-based fluorescence investigations, the dye, 8-anilino-naphthalene-1-sulfonic acrid, has been an essential marker for identification of protein MG states. It is well established that ANS binds to the MG country of the protein with higher affinity compared to that with the native and denatured states (Gussakovsky and Haas 1995). Enhanced fluorescence emission of ANS, when excited at 295 nm, has routinely been used to narrate the MG country (Stryer 1965; Semisotnov et al. 1991). It is believed that the MG intermediate states of proteins, with different structure and function, share similarities in their characteristics in terms of the content of secondary structure, hydrophobic core, and loss of tertiary structure (Kuwajima 1989; Ptitsyn 1995).

Fluorescent probes for qualitative MG state characterization

Equally mentioned in the previous section, the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid (ANS), has long been used to narrate the partially folded states of proteins (Semisotnov et al. 1991). Alongside ANS binding, native tryptophan fluorescence has been a central technique (whereby the fluorescence emission intensity of the tryptophan(s) in the protein increases differentially in the MG country compared to the native or unfolded state of the protein). This holding has been extremely useful for characterization of MG states in protein chemistry (Redfield et al. 1994).

With the availability of high-sensitivity isothermal titration calorimetry, information technology has get possible to rationalize the binding of fluorescent probes to, and the nature of the native fluorescence of, the protein MG state, by comparison these fluorescence profiles against the thermodynamic signatures reflecting associated with the binding and unfolding processes. Specific bounden of dyes to the MG state can be related to the environs of the exposed hydrophobic cadre, which is different in the MG state compared to the native and unfolded states. ANS has been used as a probe to identify molten globule states for a variety of proteins such equally homo serum albumin (HSA), recombinant man growth hormone, stalk bromelain, and more (Hawe et al. 2008). Another dye, Nile Reddish, was too used to characterize the MG states—one example involved monitoring the denaturation of HSA in the presence of GdnHCl (Hawe et al. 2008). In this study, it was observed that over the guanidine hydrochloride concentration range 0.25–1.five K, the intensity of Nile Cherry-red increased and then dropped but ANS showed maximum fluorescence intensity at ane.8 Chiliad guanidine hydrochloride which corresponds to the MG state of the protein.

In another written report, ANS and pyrene were used to characterize the MG state of BSA (Hawe et al. 2008), characterized at pH 4.two using ANS fluorescence (supplemented by CD spectroscopy, low-cal scattering, and analytical centrifugation), but pyrene showed the same intensity at pH 4.2 and pH 7.0. In some studies, bis-ANS was also tried as a probe to study the MG state of the protein. These studies indicated that MG characterization past dyes depends on the properties of a particular dye and also on the structural conformation of the molecules (Hawe et al. 2008).

Alien results on the MG country of proteins

Thermodynamic characterization of the MG state of protein has produced conflicting observations. For instance, based on the acidic pH-induced MG land of apo-α-lactalbumin, Kuwajima (1989) has reported that such a country does non undergo cooperative thermal transition to the unfolded state, which met with objections afterwards on. In the years 1991 and 1992, there were conflicting reports on the thermodynamic land of the unfolded state of the protein compared to the MG country. Xie et al. (1991) demonstrated that the guanidine hydrochloride-induced MG state of apo-lactalbumin exhibited a well-defined thermal unfolding contour based on variation of oestrus capacity by using differential scanning calorimetry (DSC). Based on their results, it was concluded that the deviation in intrinsic enthalpy betwixt the native and MG states is 32.2 kJ mol⁻¹ which is much lesser than that 133.i kJ mol^−one between its unfolded and native states (Xie et al. 1991). These observations conflicted with the findings of Ptitsyn and Kuwajima (Ptitsyn 1987; Kuwajima 1989) who proposed that the MG state of a protein does not undergo a cooperative thermal transition. The oestrus absorption bend of holo-lactalbumin obtained by Yutani et al. (1992) showed a well-defined cooperative thermal transition centered at 61.7 °C, consistent with those reported by others. However, apo-lactalbumin nether the same experimental conditions did not result in any cooperative thermal transition. Co-ordinate to the results of Yutani et al. (1992), the absence of cooperative thermal transition in DSC from MG to unfolded land clearly has a correlation with the complete loss of tertiary structure in apo-lactalbumin every bit seen in their near uv-cd spectra.

In contrast to the observations of Yutani et al. (1992), Xie et al. (1991) obtained a well-divers cooperative thermal transition in DSC centered at about 43 °C accompanied by a calorimetric enthalpy of 276 ± 13 kJ mol⁻ⁱ. Further, addition of guanidine hydrochloride upwardly to a concentration of 1.5 M showed a consistent lowering of both the transition temperature and calorimetric enthalpy. The difference between calorimetric and van't Hoff enthalpy was clearly observed by them for the MG state of α-LA indicating deviations from 2-country unfolding behavior.

Emerging qualitative data on the MG land and limited quantitative information

Recognizing difficulty in obtaining thermodynamic information on the MG state of proteins, information technology was essential to utilise alternate approaches for its characterization. One such arroyo was isothermal titration calorimetry (Hamada et al. 1994). Cytochrome c undergoes denaturation when the pH is lowered to 1.viii due to excessive repulsion of protonated residues (Goto et al. 1990). Addition of perchlorate to the acrid-unfolded state of cytochrome c pushes the poly peptide to the MG land which was monitored by using ITC. The authors obtained exothermic interaction of perchlorate with the protein, and subsequent titration of the salt into the protein led to a cooperative behavior which agreed well with the conformational transition monitored by measuring ellipticity at 222 nm. Their observations suggested that the salt-induced conformational alter from unfolded to MG state of cytochrome c tin can be approximated by 2-land transition. The authors observed the appearance of well-defined thermal transition in the protein at pH ane.viii upon addition of various amounts of NaClO₄. These observations besides conflict with the observations of Yutani et al. (1992), but agree with the result of Xie et al. (1991) that the MG state of the protein does undergo cooperative thermal transition. The striking feature of their observations is that the calorimetric enthalpy of the unfolding of the MG state determined by DSC matched well with the van't Hoff enthalpy obtained by fluorescence measurements, thereby establishing the two-state nature of the unfolding process. A nonzero, minor positive value of ΔC _p confirmed increased exposure of the cached or clustered hydrophobic groups of the poly peptide in the MG state to the solvent environment. In this report, the authors could successfully ascribe quantitative numbers to the formation of the MG state.

The germination of the MG state in acrid-unfolded cytochrome c induced by n-alkyl sulfates was also attempted and studied past using ITC (Chamani et al. 2003). Here also, the authors obtained exothermic enthalpies of conformational transition while recording the transition in parallel, using CD spectroscopy. During the same fourth dimension period, some other study described the germination of the MG state by acid-induced unfolding of cytochrome c in the presence of SDS (Moosavi-Movahedi et al. 2004). Once once more, these authors reported a well-defined thermal unfolding profile in the DSC thermogram of the MG state of cytochrome c. The major indicate of difference in their study is that the endotherm for the unfolding of the MG state was fitted by the multistate model. The claim of these authors to assign 4 energetic sub-domains in the unfolding of the MG state of cytochrome c had back up from an earlier report in literature (Fisher and Taniuchi 1992). Here, the authors did quantify the MG state to some extent, though differently than others.

Apomyoglobin has also been studied for an understanding of the MG state of the poly peptide (Hamada et al. 1995). Acid-induced (pH = two) unfolded apomyoglobin could be stabilized to the MG state by NaClO_four and Na₂SO₄. The arroyo to obtain quantitative information past employing calorimetry was the aforementioned as that used for cytochrome c. The authors found that the salt-induced conformational modify in apomyoglobin from unfolded to MG country could be approximated by a two-state transition which is exothermic in nature. The quantification of thermodynamic signatures included the possibility of estrus effects due to conformational changes in the protein. An essential point of consideration is how changes in conformation may produce the heat effects.

Partially unfolded states of human α-lactalbumin past molecular dynamics simulations have been explored by Paci et al. (2001). Based on their computational findings, they concluded that the unfolding of the MG country is not a cooperative process, thereby suggesting that the structural elements of the protein practice not unfold simultaneously.

The protein Galectin-1, in the presence of increasing concentrations of guanidine hydrochloride, exhibited a biphasic unfolding contour—attributed to the existence of at least one stable intermediate (Iglesias et al. 2003). This intermediate was argued to belong to the molten globule type, past virtue of the fact that it retained carbohydrate binding specificity. This study, withal, remained mostly qualitative with regard to the MG state; the biphasic nature of the unfolding profile suggested cooperative unfolding of the MG state to the denatured state induced by guanidine hydrochloride.

Specific quantitative information on the MG country provided past calorimetry

Equally discussed earlier, characterization of partially folded states, such every bit MG, has routinely been washed by means of fluorescence spectroscopy by using ANS as a probe (Gussakovsky and Haas 1995; Stryer 1965; Semisotnov et al. 1991). It has widely been reported that enhancement in the fluorescence emission of ANS occurs when information technology binds specifically to the partially folded states including MG states. However, this provided only qualitative information and was mainly used like a fingerprinting method. The use of ITC in characterizing the partially folded states of proteins was looked upon with great hope (Singh and Kishore 2006). It was believed that a quantitative understanding of the interaction of ANS with the MG country of the protein might be able to elucidate the mutual structural features of such an intermediate state (if any existed). Thermodynamic signatures associated with the binding of dye to the MG land, such as stoichiometry, binding constant, enthalpy change, entropy change, and van't Hoff enthalpies, tin can provide the nature of intermolecular interactions and mechanism of binding, and hence inform on the structural features of such states. Experiments were designed to written report the binding of ANS with the native state, denatured state, and MG country of the protein α-lactalbumin (Singh and Kishore 2006). The binding of ANS with the native land was observed to be weak, and the ITC-integrated heat profile of interaction with the urea-induced denatured land did not yield whatever specific binding profile. However, the ITC of the binding of ANS with the A-state (also known as the MG land) of α-lactalbumin provided a valley-shaped titration profile which followed a two-site binding model with multiple ANS molecules binding at each site. These two binding sites exhibited an exothermic interaction in the range of − 21.one kJ mol⁻¹ to − x.8 kJ mol⁻¹ and affinity constants of the lodge of 10⁴ and 10^six 1000⁻¹ at these sites, respectively (at 298.xv K). The difference of 10.3 kJ mol⁻¹ leads to a valley-shaped ITC profile instead of a normal sigmoidal ITC-integrated heat profile. The binding of ANS at these sites was also observed to take positive entropic contributions. The results suggested that each site could adapt 3.1 to xiv.five molecules which further indicates that such a binding does non occur at well-divers clefts of amino acid residues, only involves nonspecific binding which is predominantly exothermic in nature. Information technology is known that ANS usually binds to hydrophobic clusters with a possibility to also interact with ionic centers due to the presence of a sulfonate group (Gussakovsky and Haas 1995, Stryer 1965, Semisotnov et al. 1991). The data discussed above suggested dominance of polar rut effects. Nonequivalence of calorimetric and van't Hoff enthalpies associated with the binding of ANS to the MG states of proteins and absenteeism of enthalpy/entropy compensation point out the lack of well-defined binding sites on the MG state and propose the association to be nonspecific in nature (Singh and Kishore 2006).

Isothermal titration calorimetry was also employed to quantitatively sympathize the binding of ANS to the 2,2,2,-trifluorethanol (TFE)-induced MG state of concanavalin A (Banerjee and Kishore 2005). It was observed that four mol kg⁻¹ TFE at pH 2.5 is able to induce the MG state in concanavalin A which was well characterized past fluorescence and circular dichroism spectroscopies. The DSC thermal profiles of concanavalin A at pH v.two, where the protein does not be in the MG land in the absenteeism and presence of TFE, exhibited a well-defined cooperative transition to the unfolded state. Notwithstanding, the MG state of lectin at pH ii.iv lacked such cooperative thermal transition. Hither also, the binding of ANS to the MG state of concanavalin A followed a ii-state bounden model with values of binding constants of the order of 10³ and 10^v K⁻¹. Values for the enthalpy of bounden ranged from − 0.20 to − xx.27 kJ mol⁻¹ at 298.15 K. It is worth noticing hither that the association of ANS with the MG land of concanavalin A is also observed to be exothermic in nature with favorable entropy alter. The stoichiometry of bounden at each site is more than one, varying from three.8 to 24.8, suggesting a larger expanse of the binding site.

Studies take also been done with the partially folded country of α-lactalbumin induced past the mixture of hexafluoroisopropanol and guanidinium thiocyanate, the former being a helix inducer and the latter, a denaturant (Sharma and Kishore 2008). ANS binding to these partially folded states with varying levels of secondary and tertiary structures also followed a ii-type binding site model, with the lodge of binding constants as 10^two to x^iv M⁻¹, but weak endothermic enthalpy and favorable entropy change. These intermediate states exhibited thermal transition to the unfolded state, in agreement with the data provided past absorbance changes.

Binding of ANS to the TFE-induced partially folded states of myoglobin was studied by using calorimetry and spectroscopy (Talele and Kishore 2015). Hither, ane.v mol kg⁻¹ TFE was sufficient to induce the MG state in the poly peptide. The DSC thermal profile did not bear witness whatsoever cooperative thermal transition, and ITC studies on the bounden of ANS with the TFE-induced MG land of myoglobin yielded thermodynamic parameters of binding according to a two-site bounden model. The binding constants obtained hither were of the order of 10^v and 10⁷ M^−one, with exothermic enthalpy of binding –(xviii.08 ± 0.14) kJ mol⁻¹ and –(1.04 ± 0.05) kJ mol⁻¹, respectively, at 298.15 K. Consequent with earlier observations, the binding of ANS with the MG state of myoglobin was also observed to be enthalpy driven. Surfactants which induced a partially folded state of α-LA likewise offered two sequential binding sites of ANS molecules with binding constants of the social club of 10³ and 10^iv and exothermic enthalpy of bounden of –(12.1 ± 0.03) kJ mol^−one and –(35.7 ± i.five) kJ mol⁻¹, thereby establishing consistency of the binding model fitted to the experimental data (Misra and Kishore 2011).

Even though qualitative information on the MG states has emerged over time, the determinants of MG stability and the extent of specific packing in the MG state are notwithstanding a thing of investigation. Germination of a molten globule country at acidic pH has been demonstrated for the periplasmic binding proteins [lipopolysaccharide-binding protein (LBP), leucine-isoleucine-valine-binding poly peptide (LIVBP), maltose-binding poly peptide (MBP), and retinol-binding protein (RBP)] (Prajapati et al. 2007). These proteins were shown to possess the power to demark to their corresponding ligands without undergoing transition to the native state, albeit with a smaller affinity in near cases (Prajapati et al. 2007). These observations suggested that periplasmic bounden proteins preserve a significant degree of long-range order fifty-fifty in the MG land. The DSC thermograms of all these four proteins exhibited well-defined unfolding behavior, and the thermal stability of the proteins could be raised by almost 15 °C upon bounden their cognate ligand. Further, all their transitions fitted well to the two-state unfolding model. Here, the authors observed an most complete loss of tertiary structure of the proteins, but however appreciable endotherm in the DSC profiles and pregnant binding to the corresponding ligands.

Electric current agreement suggests that MG states can exhibit varying degrees of compactness and solvent accessibility (Dijkstra et al. 2018). Information technology is besides argued that the nature of the MG country is highly sequence dependent and that the heat capacity versus temperature curves may showroom heat-induced MG states. These authors farther propose that the MG state does not necessarily adopt a detail conformation and that natively disordered proteins can exhibit multiple MG-similar states. With regard to the nature of DSC thermal profiles, the authors argue that MG states do testify peaks in the heat capacity curves with a reduced enthalpy to that of unfolding of the native protein. For the protein staphylococcal nuclease, 3 different partially folded intermediates lacking rigid construction, simply nevertheless containing significant tertiary construction, were observed in anion-induced refolding experiments (Uversky et al. 1998). Although more often than not qualitative in nature, that report suggested that the intermediates observed represent the equilibrium counterparts of transient kinetic intermediates.

Pressure perturbation calorimetry (PPC) has besides been applied to characterize the molten globule country of cytochrome c at pH four.5. This technique evaluates the temperature dependence of the thermal expansion coefficient of the protein (Nakamura and Kidokoro 2012). The thermal unfolding curve obtained past PPC could exist fitted by iii-state assay including the MG state. The partial specific volume of the MG state was institute to be in between that of the N and D states. Hither, the cooperative thermal transition obtained from the MG to the unfolded state is not based on oestrus capacity but based on volumetric parameters determined on DSC. Even though the observed thermodynamic quantity determined here is somewhat nonstandard, information technology demonstrated that cytochrome c in the MG country does exhibit sufficient compactness which upon heating yields a cooperative thermal transition. The alter in volume of the protein every bit a event of conformational transitions is small and may be either positive or negative. Information technology has been observed that the change in solution book, per unit volume of the protein, for the unfolding of ribonuclease A, ubiquitin, lysozyme, and eglin C, all converge to a common value at high temperature (Schweiker et al. 2009). The protein cytochrome c exhibited a unlike value of this volumetric ratio than the higher up mentioned proteins. This difference was assigned to a loosely packed structure of cytochrome c compared to the other proteins. Similar differences are seen in the thermal unfolding behavior of the MG states of cytochrome c and the other mentioned proteins where the thermal transition for the former protein is cooperative and for the latter gear up of proteins is noncooperative (Dolgikh et al. 1985; Nakamura et al. 2007, 2011; Potekhin and Pfeil 1989; Hamada et al. 1994). The volume of the intermediate state of cytochrome c is observed to be smaller than that of the denatured land which indicates that the hydrophobic cadre in the protein is nevertheless retained in the MG land of the protein (Nakamura and Kidokoro 2012).

Isothermal titration calorimetry was used to determine the unfolding enthalpy of the pH 4 induced MG state of apomyoglobin (Tyagi et al. 2009). In that experiment, apomyoglobin at pH five.5 was placed in the cell of the ITC and HCl was sequentially titrated to determine the heat of interaction. Later on correcting for the oestrus effects associated with protonation of 22 carboxylate residues, the enthalpy change associated with the unfolding of apomyoglobin to the unfolded state of the poly peptide is zero, and on this footing, the authors argued against cooperative unfolding of the MG state. These observations are consistent with the results obtained by Griko and Privalov (1994). In that location accept been other reports on apomyoglobin describing very shallow thermal unfolding curves for the MG state (Nishii et al. 1995). In agreement with the above ITC observations, Hamada and coworkers (Hamada et al. 1995) reported a zero enthalpy of anion-induced folding of equus caballus heart myoglobin at ten °C.

Calcium-bounden lysozyme [canine milk lysozyme (CML)] has also been studied using DSC. CML exhibits ii thermal transitions which represent to the native to intermediate and intermediate to unfolded transitions, respectively (Koshiba et al. 2001). In contrast to the absenteeism of a thermal transition in apo-α-LA, the intermediate land of CML showed a well-defined oestrus absorption superlative. An interesting feature of this transition was that it occurred at a temperature college than that of the offset transition. Whether or non the transition of an MG state to the unfolded state is cooperative was previously suggested to be related to the extent of unique tertiary structure of the protein (Privalov 1979; Privalov and Gill 1988; Shakhnovich and Finkelstein 1989). Therefore, any deviations in the thermal unfolding contour of an MG state must reflect the unique packing of the amino acid residues in such a state. This in turn should be strongly dependent on the nature of the amino acid sequence (Koshiba et al. 2000).

A qualitative study on the acrid-induced MG country of the prion protein was reported as information technology was believed that insights into the conformational aspects of the intermediate may provide crucial insights into the mechanism of oligomerization and pathogenic conversion, thereby helping in the design of new medical chaperones useful in the treatment of prion diseases (Honda et al. 2014). However, that study was largely qualitative and did not address any thermodynamic aspects. In this direction, the MG state of bovine pancreatic trypsin inhibitor was besides addressed (Ferrer et al. 1995). The difference in the cooperativity of transition was correlated with the extent of α-helical or β-sheet structure in the protein.

The unfolding of Northward-acylamino acid amido hydrolase (aminoacylase) has been investigated in the presence of aspartate using ANS fluorescence spectra, CD, enzyme activity, and intrinsic fluorescence emission spectra. Previous studies on the denaturation of aminoacylase using urea, guanidine hydrochloride, SDS, and temperature induction methods showed a two-state transition without whatever indication of intermediates (Rariy and Klibanov 1997; Sato et al. 1996). Later on, Bai et al. (1999) showed the existence of an intermediate state in the guanidine hydrochloride-induced unfolding of aminoacylase. Aspartate, a weak acid with pI = 2.77, was used to study the unfolding of aminoacylase (Xie et al. 2003) due to the fact that information technology helps to maintain the structural integrity of the poly peptide and only weakly affects the enzyme functions (information technology also helps to keep the dimeric structure of the protein intact). The pH-dependent fluorescence transition of aminoacylase in the presence of aspartate indicated two distinct processes. The transition from pH five.6 to four.0 showed a cerise shift, followed past a blue shift from pH 3.vii to 3.0. This indicated a structural modification of the protein, in which the tryptophan moiety is surrounded more than by hydrophobic residues. The CD results reinforced the fluorescence observations in the sense that an increment in aspartate concentration acquired the secondary structure of aminoacylase to revert to the native-like state (merely with an increase in ANS fluorescence intensity). This indicated the presence of an MG country during the unfolding of aminoacylase.

The anionic surfactant, sodium dodecylsulfate, has been reported to induce a MG state in the highly negatively charged ferricytochrome c at pH 12.eight (Jain et al. 2018). Of note here, even though both the protein and head groups of the surfactant molecules in the pre-micellar state carry a negative charge, the stabilization of the unfolded land to the MG land is still observed. In this report, the MG state was reported to have a native-like α-helical structure but lacked an appreciable third structure. SDS-induced unfolding of the protein studied by CD and fluorescence spectroscopies provided a standard molar Gibbs free energy change of xi.3 kJ mol^−ane and 8.four kcal mol⁻¹, respectively, at 298.xv Thou. Though positive, these values are relatively pocket-size, and therefore propose a lack of cooperativity in the unfolding of the MG state to an unfolded state. The authors attribute preferential stabilization of the MG state to interactions between Na⁺ ions and the negatively charged poly peptide and hydrophobic interactions via the tail groups of the surfactant. It was farther inferred that sub-micellar concentration of SDS leads to enhancement of thermal stability and prevents common cold denaturation of the alkali-induced denatured country of cytochrome c. The information bachelor from this study reflected more on the mechanistic details but suggested lack of cooperativity in the transition from native to MG state.

Maltose-binding protein (MBP) is a periplasmic unmarried-concatenation monomeric protein of Escherichia coli having 370 amino acids and no disulfide bonds. MBP forms a MG country at pH 3. The value of ΔC _p associated with the thermal unfolding of a protein tin can provide information on the extent of compactness of the protein. The unfolding of salt-induced MG state of yeast iso-i-cytochrome c exhibited ΔC _p = 3.two kJ K⁻ⁱ mol⁻¹; for equine-cytochrome c, the value was in the range of 1.6 kJ K⁻¹ mol^−ane to 2.4 kJ K⁻¹ mol⁻¹, values which are lower than the alter in the heat chapters of the protein undergoing transition from native to denatured state (Kuroda et al. 1992; Hamada et al. 1994). The ΔC _p value for the unfolding of the MG state of MBP was observed to be only 30% dissimilar than the native state of the poly peptide. This report adult a quantitative understanding of the MG state of MBP in terms of modify in oestrus chapters and its correlation to the accessible hydrophobic surface area (Sheshadri et al. 1999).

Contempo qualitative studies on the MG land

A number of interesting, largely qualitative, studies on the MG state accept been recently published, and some of these examples are discussed ahead. Stabilization of apo-α-lactalbumin by binding of epigallocatechin-3-gallate has been observed both with the native and MG states of the quondam. Bounden of epigallocatechin gallate with the MG state of the poly peptide indicated sufficient extent of structural features in the intermediate country to allow bounden to the incoming ligand molecules (Radibratovic et al. 2019). Formation of the MG land in homodimeric CcD B [controller of cell death B] protein has been characterized spectroscopically (Baliga et al. 2019). That study provided insights into structural and dynamic properties of a depression-pH state of CcD B. Several other reports addressing either the germination or role of the MG state accept been published (Peixoto et al. 2019, Kozak et al. 2018, Kulkarni and Uversky 2018; Uversky 2018; Wirtz et al. 2018; Samanta et al. 2017, Ithychanda et al. 2017).

Force per unit area perturbation calorimetry was used to sympathize the MG state of cytochrome c, finding a high-temperature reversible oligomerization process (Zhang et al. 2017). Here, the transition from MG state to unfolded state was studied at different concentrations of the protein. The calorimetric information suggested at to the lowest degree a six-state unfolding process involving 2 MG states, the denatured state and further arrangement of the denatured state to dimeric, trimeric, and tetrameric forms, with each step being reversible. The cooperative thermal transition of the MG state of cytochrome c was also observed hither. Xie et al. (1991) have reported that at neutral pH, the enthalpy of the unfolded state of α-LA differs past 100.8 kJ mol^−ane at 25 °C from its MG state. Yutani et al. (1992), who observed that the enthalpy difference betwixt the MG state and the presumed unfolded country is almost nothing, pointed out this difference to the model used past Xie et al. (1991). They assigned this discrepancy to the values of enthalpy alter for protein denaturation and bounden of denaturant with the protein. It must exist noted here that the MG state of α-LA obtained by Xie et al. was induced by GdnHCl denaturant, whereas that addressed by Yutani et al. did not involve whatsoever denaturant. A year subsequently the report of Yutani et al. virtually enthalpic equivalence of the MG and unfolded state of α-LA, Xie et al. (1993) argued that the decision of enthalpic equivalence of these states is incorrect and assigned the absenteeism of thermally induced transition to ionic strength dependence. It was further argued that in improver to enthalpy change, entropy change between states can also account for the observed departure.

To conclude this department, nosotros annotation that calorimetry has contributed significantly in the current knowledge of the determinants of protein structure (Ladbury and Doyle 2004; Velicelebi and Sturtevant 1979). A major contribution to this understanding has come up from both differential scanning calorimetry (Privalov and Dragan 2007; Mazurenko et al. 2017) and isothermal titration calorimetry (ITC) (Carra et al. 1996; Singh and Kishore 2006).

Conclusions and time to come perspectives

Equilibrium intermediate states, such equally the MG, have resemblance to the kinetic intermediate states during the protein folding procedure. As discussed above and is evident from the Fig.ii, much endeavour has been defended towards elucidating a detailed understanding of MG and other partially folded states. It is likewise articulate that bulk of the efforts take yielded qualitative information; notwithstanding, the quantitative information has been very express. In such a scenario, awarding of loftier-sensitivity ITC and DSC has provided express, merely useful, quantitative information about the backdrop of the MG state. All the same, the absenteeism or presence of cooperative thermal transition in going from the MG state to the unfolded state remains unclear (Fig. ii). Some studies, based upon calorimetry, have indicated that equilibrium MG states of a wide variety of proteins share common structural features based upon their exhibition of similar thermodynamic signatures accompanying the bounden of ANS. These common structural features take suggested two sets of nonspecific binding sites for ANS on the MG state of proteins which could be the combination of hydrophobic and ionic sites (as the former possesses both types of molecular properties). The thermodynamic parameters accompanying the binding of ANS with the MG states of unlike proteins advise an exothermic nature to the binding, with significant desolvation as reflected by positive values of the change in entropy. We even so do not have thermodynamic signatures accompanying the processes shown in Fig. 2 for a large number of proteins which could lead to deriving general guidelines well-nigh thermodynamic states of such intermediates. Isothermal titration calorimetry, used in combination with differential scanning calorimetry, offers great potential for providing the required insights.

An external file that holds a picture, illustration, etc. Object name is 12551_2019_527_Fig2_HTML.jpg

Scheme describing known methods to generate the MG state of a poly peptide and questions to which answers are nonetheless non clear

Footnotes

Publisher's note

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Contributor Information

Eva Judy, Electronic mail: ni.ca.btii@ydujave.

Nand Kishore, Electronic mail: ni.ca.btii.mehc@kdnan.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557940/