Thermostability of the N-Terminal RNA-Binding Domain of the SARS-CoV Nucleocapsid Protein: Experiments and Numerical Simulations

Differential scanning calorimetry, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and numerical simulations were used to study the thermostability of the N-terminal RNA-binding domain (RBD) of the SARS-CoV nucleocapsid protein. The transition temperature of the RBD in a mixing buffer, composed of glycine, sodium acetate, and sodium phosphate with 100 mM sodium chloride, at pH 6.8, determined by differential scanning calorimetry and circular dichroism, is 48.74°C. Experimental results showed that the thermal-induced unfolding-folding transition of the RBD follows a two-state model with a reversibility >90%. Using a simple Gō-like model and Langevin dynamics we have shown that, in agreement with our experiments, the folding of the RBD is two-state. Theoretical estimates of thermodynamic quantities are in reasonable agreement with the experiments. Folding and thermal unfolding pathways of the RBD also were experimentally and numerically studied in detail. It was shown that the strand β₁ from the N-terminal folds last and unfolds first, while the remaining β-strands fold/unfold cooperatively.     

On the origins of the weak folding cooperativity of a designed ββα ultrafast protein FSD-1

FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5), we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K) of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K) as well as the denatured structures.     

Equilibrium structure and folding of a helix-forming peptide: circular dichroism measurements and replica-exchange molecular dynamics simulations

We have performed experimental measurements and computer simulations of the equilibrium structure and folding of a 21-residue alpha-helical heteropeptide. Far ultraviolet circular dichroism spectroscopy is used to identify the presence of helical structure and to measure the thermal unfolding curve. The observed melting temperature is 296 K, with a folding enthalpy of -11.6 kcal/mol and entropy of -39.6 cal/(mol K). Our simulations involve 45 ns of replica-exchange molecular dynamics of the peptide, using eight replicas at temperatures between 280 and 450 K, and the program CHARMM with a continuum solvent model. In a 30-ns simulation started from a helical structure, conformational equilibrium at all temperatures was reached after 15 ns. This simulation was used to calculate the peptide melting curve, predicting a folding transition with a melting temperature in the 330-350 K range, enthalpy change of -10 kcal/mol, and entropy change of -30 cal/(mol K). The simulation results were also used to analyze the peptide structural fluctuations and the free-energy surface of helix unfolding. In a separate 15-ns replica-exchange molecular dynamics simulation started from the extended structure, the helical conformation was first attained after approximately 2.8 ns, and equilibrium was reached after 10 ns of simulation. These results showed a sequential folding process with a systematic increase in the number of hydrogen bonds until the helical state is reached, and confirmed that the alpha-helical state is the global free-energy minimum for the peptide at low temperatures.     

Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study

A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of alpha-synuclein (alphaS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of alphaS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of alphaS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the alphaS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of alphaS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes.     

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.     

Energetics of MazG Unfolding in Correlation with Its Structural Features

MazG is a homodimeric α-helical protein that belongs to the superfamily of all-α NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-α NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual α-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH < 6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein–DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.     

Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction

Background

The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results.

Methodology/Principal Findings

Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines.

Conclusion/Significance

From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.     

Thermodynamic effects of mutations on the denaturation of T4 lysozyme.

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.     

Folding energetics of a multidomain protein, flagellin.

Thermodynamic investigations of flagellin from Salmonella typhimurium and its proteolytic fragments were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements. A new method of analysis for a multi-state transition based on our original theoretical treatment of thermodynamic equations has been developed to analyze those data. The analysis of DSC curves confirmed the three thermodynamic domains of flagellin. The thermodynamic parameters of each domain were revised from those previously reported and the new values of the parameters have a good correlation to the apparent molecular masses of the morphological domains. CD melting measurements at far and near-UV wavelengths showed sequential unfolding of the domains. Therefore, we could reasonably assign the thermodynamically identified domains to the morphological domains. Further analysis of both DSC and CD data provided insights into the folding energetics of the multidomain structure of flagellin. An inner domain (Df1) of flagellin in the filament unfolds through a relatively broad transition, while the two outer domains unfold cooperatively and show sharp transitions. This indicates that the interdomain interactions between Df1 and D2 has different characteristics from the apparently more intimate interactions between D2 and D3. These characteristics suggest that flagellin is organized with relatively flexible domains and rigid domains, which appears to be responsible for the well-regulated assembly mechanism of the bacterial flagellar filament.     

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.     

Osmotically induced helix-coil transition in poly(glutamic acid)

Protein folding and conformational changes are influenced by protein-water interactions and, as such, the energetics of protein function are necessarily linked to water activity. Here, we have chosen the helix-coil transition in poly(glutamic acid) as a model system to investigate the importance of hydration to protein structure by using the osmotic stress method combined with circular dichroism spectroscopy. Osmotic stress is applied using poly(ethylene glycol), molecular weight of 400, as the osmolyte. The energetics of the helix-coil transition under applied osmotic stress allows us to calculate the change in the number of preferentially included water molecules per residue accompanying the thermally induced conformational change. We find that osmotic stress raises the helix-coil transition temperature by favoring the more compact α-helical state over the more hydrated coil state. The contribution of other forces to α-helix stability also are explored by varying pH and studying a random copolymer, poly(glutamic acid-r-alanine). In this article, we clearly show the influence of osmotic pressure on the peptide folding equilibrium. Our results suggest that to study protein folding in vitro, the osmotic pressure, in addition to pH and salt concentration, should be controlled to better approximate the crowded environment inside cells.     

Thermal unfolding of calreticulin. Structural and thermodynamic characterization of the transition

Calreticulin (CRT) is a calcium-binding protein that participates in several cellular processes including the control of protein folding and homeostasis of Ca²⁺. Its folding, stability and functions are strongly controlled by the presence of Ca²⁺. The oligomerization state of CRT is also relevant for its functions. We studied the thermal transitions of monomers and oligomers of CRT by differential scanning calorimetry (DSC), circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the presence and absence of Ca²⁺. We found three and two components for the calorimetric transition in the presence and absence of Ca²⁺ respectively. The presence of several components was also supported by CD and FTIR spectra acquired as a function of the temperature. The difference between the heat capacity of the native and the unfolded state strongly suggests that interactions between protein domains also contribute to the heat uptake in a calorimetry experiment. We found that once unfolded at high temperature the process is reversible and the native state can be recovered upon cooling only in the absence of Ca²⁺. We also propose a new simple method to obtain pure CRT oligomers.     

Stability and folding studies of the N-domain of troponin C. Evidence for the formation of an intermediate

We report here on the stability and folding of the 91 residue alpha-helical F29W N-terminal domain of chicken skeletal muscle troponin C (TnC(1-91)F29W), the thin filament calcium-binding component. Unfolding was monitored by differential scanning calorimetry, circular dichroism, and intrinsic fluorescence spectroscopy using urea, pH, and temperature as denaturants, in the absence and in the presence of calcium. The unfolding of TnC(1-91)F29W was reversible and did not follow a two-state transition, suggesting that an intermediate may be present during this reaction. Our results support the hypothesis that intermediates are likely to occur during the folding of small proteins and domains. The physiological significance of the presence of an intermediate in the folding pathway of troponin C is discussed.     

DSC deconvolution of the structural complexity of c-MYC P1 promoter G-quadruplexes

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P₁ promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K⁺] and 130 mM [K⁺]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).     

The Herpes Simplex Virus Triplex Protein, VP23, Exists as a Molten Globule

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.     

Free cysteine modulates the conformation of human C/EBP homologous protein

The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid L-cysteine. Addition of L-cysteine caused a significant dose-dependent decrease in the protein helicity--dropping from 69.1% to 23.8% in the presence of 1 mM of L-cysteine--and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 μM, an 8:1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free L-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of L-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress.