Unfolding Transition Research Articles

Melting of DNA Nonoriented Fibers: A Wide-Angle X-ray Diffraction Study

The melting transition of A- and B-DNA has been investigated by wide-angle X-ray diffraction. A significant crystalline phase is present in both the systems, even if the fibers have not been artificially aligned. The behavior of the intramolecular Bragg peaks of both A- and B-DNA as a function of the temperature clearly reveals the unfolding structural transition of the double helix. This transition occurs at the same temperature as the melting of the crystalline phase. The trends of the intramolecular correlations and the index of crystallinity are nicely described by the Peyrard-Bishop-Dauxois model for DNA melting. A description of the processes taking place at a microscopic level, i.e., double-helix deformation, crystalline dilation, and collapse, on approaching and during thermal melting is proposed.

Protein Unfolding Through Nanopores

In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.

Synthesis and Single-Chain Folding of Amphiphilic Random Copolymers in Water

Amphiphilic random methacrylate copolymers, consisting of poly(ethylene glycol) (PEG) and alkyl pendent groups, undergo reversible single-chain self-folding in water via intramolecular hydrophobic interaction, to generate a dynamic unimolecular hydrophobic nanospace, similar in shape but structurally different relative to micelles and microgel star polymers. These copolymers were prepared by the ruthenium-catalyzed living radical copolymerization of a PEG methacrylate (PEGMA) and an alkyl methacrylate (RMA; R, −CnH2n+1, n = 1–18), where copolymer composition, degree of polymerization, and hydrophobic R moiety were varied. Detailed structural and chain-folding characterization has revealed: single-chain folding is favored with the RMA content 20–40 mol % per chain; the hydrophobic inner compartment (or the self-folded structure) is stable even at a high polymer concentration (up to ∼6 wt %); and folded–unfolded transition occurs on addition of methanol or by elevating solution temperature, finally to phase...

Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.

Preferential Water Exclusion in Protein Unfolding

Association of water with protein plays a central role in the latter's folding, structure acquisition, ligand binding, catalytic reactivity, oligomerization, and crystallization. Because these phenomena are also influenced by the net charge content on the protein, the present study examines the association of water with cytochrome c held at different pH values so as to allow its side chains to ionize to variable extents. Equilibrium unfolding of differently charged cytochrome c molecules in water-methanol binary mixtures, where the alcohol acts as the cosolvent denaturant, was used to quantify the preferential exclusion of water during the unfolding transition. The extent of exclusion was found to be related to the net-charge-dependent molecular expansion of the protein in an alcohol-free aqueous medium. The degree of water exclusion was also found to be linearly related to the observed rate of protein unfolding, where the net charge contents of the initial and final states are the same. The results suggest that side-chain ionization, molecular expansion due to charge repulsion, and hence the loss of tertiary contacts lead to additional water-protein association. Protein unfolding rates appear to be linearly correlated with the effective number of water molecules excluded across the end states of unfolding equilibria.

High Throughput Screening of Formulations to Optimize the Thermal Stability of Therapeutic Monoclonal Antibody

Monoclonal antibodies (mAb) are progressively more important class of biotherapeutics. Antibodies are heterodimers composed of two heavy and two light polypeptide chains linked by disulfide bonds. The majority of recombinant antibodies currently under development are IgGs. Successful development of therapeutic antibodies depends not only on attainment of desired biological activities but also on physicochemical properties of these molecules, such as homogeneity, monomer oligomeric state and stability enabling manufacturing, storage and delivery. Antibody stability is affected by its formulation: ionic strength, pH, buffering substance, presence of excipients and protein concentration. Among many techniques used to study the effects of formulation on stability of antibodies, differential scanning fluorimetry (DSF) is distinguished by an unprecedented throughput and minimal material consumption. DSF measures the midpoint of unfolding transition of protein - Tm - during thermal denaturation based on the change in fluorescence intensity of environmentally sensitive dye Sypro Orange.Here, we analyzed the impact of formulation on stability of mAb1 using DSF adapted to the 96-well plate format. Two to three thermal unfolding transitions were visible for mAb1, which reproduced closely the melting profile obtained with differential scanning calorimetry (DSC). Good correlation was also observed between Tm of the main thermal unfolding transitions determined by DSF and DSC, with the former being on average lower by 3°C. Basal fluorescence of Sypro Orange was enhanced by the presence of detergents, limiting the use of this approach to the diluted detergent solutions. We have shown that low pH or high salt concentrations decreases the thermal stability of mAb1, whereas some excipients such as sucrose, methionine and phosphate buffer increase its stability. We have also shown that the throughput of DSF can be increased further with the use of 384-well plate.

Difference FT-IR Studies on the Effects of Buffers on Nucleotide Binding to Reca

The Escherichia coli protein RecA catalyzes DNA strand exchange and plays a role DNA repair and genetic recombination. Nucleotide binding influences RecA oligomerization and its affinity for DNA. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. Past circular dichroism (CD), infrared (IR), and fluorescence studies suggest only minimal buffer dependent changes in nucleotide binding and secondary structure that did not explain the large buffer dependent differences in RecA stability and unfolding profiles. These observations led to further investigations of how the four common biological buffers Tris, HEPES, MES, and PO4 alter RecA structure and nucleotide binding. Here we have employed difference infrared spectroscopy utilized in conjunction with caged nucleotides to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. These higher resolution studies are aimed at detecting if the buffers alter nucleotide binding to RecA. Preliminary results show that ADP binding results in perturbations in Gln, Glu, Asp, Asn, Tyr, and Lys residues and secondary structural changes. Initial comparisons of difference spectra obtained in the four buffers show some similar changes but also show some differences. These differences between RecA-ADP minus RecA difference spectra will be discussed.

Effect of G-Quadruplex Stabilizing Compound on the Folding and Unfolding Pathway of Human Telomeric DNA

We use an optical tweezers platform to study the folding and unfolding pathway of individual molecules containing single-stranded DNA human telomeric G-quadruplex (G4) sequence, (TTAGGG)4. In the presence of 150 mM Na+ solution, these DNA molecules are folded into G-quadruplex structure based on the Hoogsteen basepairing. When forces were applied to unfold the G4-containing DNA molecules, most of the unfolding traces show one or two unfolding transitions. The refolding process also shows one or two refolding steps, suggesting the existence of a stable intermediate unfolding/refolding state. The total unfolding distance is about 8.4 nm, consistent with the expected unfolded G4 structure. However, when the DNA molecules were pre-incubated with a G4 stabilizing ligand BMVC (3,6-bis(1-methyl-4-vinylpyridinium) carbazole), most DNA molecules showed three unfolding and refolding transitions, suggesting of two unfolding/refolding intermediate states. Our observation suggests that BMVC-bound G4 structures unfold and refold in two unfolding/refolding intermediates, in contrast of the one in the ligand-free G4 structure.

Dcm Mutations in Tropomyosin Induce Loss of Thermal Stability When the Mutation is Present in a Single Chain of the Tm Dimer

Individuals carrying cardiomyopathy mutations normally have the mutation in one of the two genes and therefore both wt and mutant proteins will be present in the cell. In the case of dimeric protein then homo and heterodimers are likely to exist side by side. We have made α-tropomyosin (Tm) in which one or both chains of the dimer carry a single point mutation associated with dilated cardiomyopathy (DCM). The mutations are E40K, E54K and D230N. Protein unfolding was monitored for a 7 μM solution of Tm (0.5 M KCl, 5 mM MgCl2, 1 mM DTT, 20 mM KPi buffer pH 7.0) using the CD signal at 222 nm with a constant heating rate of 1 °C min−1. The unfolding profile was fully reversible and repeated unfolding curves were superimposable. Each Tm unfolded in via 3 unfolding transitions; those for wt Tm occurred at 40, 47 & 53 °C. The presence of a mutation in a single chain caused a loss of thermal stability with significant unfolding at 37 °C. The least stable transition occurred with a mid-point at 3-5 degrees lower temperature for each of the three Tms carrying a single mutation. The other two transitions were similar to wt for all three constructs. When both chains carried the mutation, the all three transitions were similar to those of the wt Tm. Supported by the Wellcome Trust and a University of Kent Studentship.

Effect of interactions with the chaperonin cavity on protein folding and misfolding.

Recent experimental and computational results have suggested that attractive interactions between a chaperonin and an enclosed substrate can have an important effect on the protein folding rate: it appears that folding may even be slower inside the cavity than under unconfined conditions, in contrast to what we would expect from excluded volume effects on the unfolded state. Here we examine systematically the dependence of the protein stability and folding rate on the strength of such attractive interactions between the chaperonin and substrate, by using molecular simulations of model protein systems in an idealised attractive cavity. Interestingly, we find a maximum in stability, and a rate which indeed slows down at high attraction strengths. We have developed a simple phenomenological model which can explain the variations in folding rate and stability due to differing effects on the free energies of the unfolded state, folded state, and transition state; changes in the diffusion coefficient along the folding coordinate are relatively small, at least for our simplified model. In order to investigate a possible role for these attractive interactions in folding, we have studied a recently developed model for misfolding in multidomain proteins. We find that, while encapsulation in repulsive cavities greatly increases the fraction of misfolded protein, sufficiently strong attractive protein-cavity interactions can strongly reduce the fraction of proteins reaching misfolded traps.

Coupling between Protein Conformation and Local Unfolding Highlights the Role of Disorder in Protein Function and Suggests a New Target for Tuberculosis Treatment

Mycobacterium tuberculosis protein tyrosine phosphatase B (PtpB), which plays a key role in tuberculosis virulence, protects its active site from oxidation through a large-amplitude conformational change. The proposed regulatory mechanism for this protective motion involves a local unfolding event. We quantitatively show that local unfolding transitions can be coupled to other conformational changes in proteins. Molecular dynamics simulations were used to determine the conformations of a disordered protein region and the energy landscape for local unfolding. The accuracy of this energy landscape was tested and confirmed experimentally, providing rigorous support for our simulation approaches. Our work demonstrates that local unfolding can depend on protein conformation by changes in the stability of the disordered region. These results provide a mechanism for the internal regulation of protein conformational changes, which highlights new targets for the development of tuberculosis therapeutics.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Effects of pH and buffer concentration on the thermal stability of etanercept using DSC and DLS.

The protein size, electrical interaction, and conformational stability of etanercept (marketed as Enbrel®) were examined by thermodynamic and light scattering methods with changing pH and buffer concentration. As pH of etanercept increased from pH 6.6 to 8.6, electrical repulsion in the solution increased, inducing a decrease in protein size. However, the size changed less in high buffer concentration and irreversible aggregation issues were not observed; in contrast, aggregates of about 1000 nm were observed in low buffer concentration at the pH range. Three significant unfolding transitions (Tm) were observed by differential scanning calorimetry (DSC). Unlikely to Tm1, Tm2 and Tm3 were increased as the pH increased. Higher Tm at high buffer concentration was observed, indicating increased conformational stability. The apparent activation energy of unfolding was further investigated since continuous increase of Tm2 and Tm3 was not sufficient to determine optimal conditions. A higher energy barrier was calculated at Tm2 than at Tm3. In addition, the energy barriers were the highest at pH from 7.4 to 7.8 where higher Tm1 was also observed. Therefore, the conformational stability of protein solution significantly changed with pH dependent steric repulsion of neighboring protein molecules. An optimized pH range was obtained that satisfied the stability of all three domains. Electrostatic circumstances and structural interactions resulted in irreversible aggregation at low buffer concentrations and were suppressed by increasing the concentration. Therefore, increased buffer concentration is recommended during protein formulation development, even in the earlier stages of investigation, to avoid protein instability issues.

Kinetics and conformational stability studies of recombinant leucine aminopeptidase

Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase (rLAP) to explain the gain of activity observed when incubated at 37°C after its production. Unfolding transitions of rLAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8±0.4 and 3.7±0.4kcalmol−1, respectively. Thermal stability conformation of rLAP is 2.6±0.1 and 6.1kcalmol−1 for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with l-leucine-p-nitroanilide (L-pNA) as substrate. The catalytic efficiency was 3.87±0.10min−1μM−1 at 37°C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and rLAP; however rLAP is selective and specific to remove N-terminal groups from amino acids.

Critical Evaluation of the Two-State Model Describing the Equilibrium Unfolding of the PI3K SH3 Domain by Time-Resolved Fluorescence Resonance Energy Transfer

It appears that equilibrium unfolding transitions of many small proteins can be described as two-state transitions, because the probes commonly used to measure such transitions cannot detect the underlying heterogeneity inherent in protein folding and unfolding reactions. Time-resolved fluorescence or Forster resonance energy transfer (TRFRET) measurements have the potential to uncover such heterogeneity and to test the cooperativity of protein folding reactions. Here, TRFRET measurements have been used to study the equilibrium unfolding of the SH3 domain of PI3 kinase. The single tryptophan residue (W53) was used as the FRET donor, and a covalently attached thionitrobenzoate moiety at either of two sites (C17 and C70) was used as the FRET acceptor. The individual lifetime and amplitude components estimated from fitting the fluorescence decay kinetics to the sum of three or four exponentials were determined over a range of denaturant concentrations. The equilibrium unfolding transitions reported by these components were found to be noncoincident, suggesting the presence of multiple conformations in equilibrium during the course of unfolding. Fluorescence lifetime distributions were also generated by the model-free maximum entropy method of analysis. Different segments of the protein were found to show differences in the expansion of the native state at low denaturant concentrations, suggestive of gradual structural transitions. The unfolded protein was found to swell at increasingly high denaturant concentrations. The evolution of the fluorescence lifetime distributions with increasing denaturant concentration was also found to be incompatible with a two-state equilibrium unfolding model.

Spatio-temporal hierarchy in the dynamics of a minimalist protein model

A method for time series analysis of molecular dynamics simulation of a protein is presented. In this approach, wavelet analysis and principal component analysis are combined to decompose the spatio-temporal protein dynamics into contributions from a hierarchy of different time and space scales. Unlike the conventional Fourier-based approaches, the time-localized wavelet basis captures the vibrational energy transfers among the collective motions of proteins. As an illustrative vehicle, we have applied our method to a coarse-grained minimalist protein model. During the folding and unfolding transitions of the protein, vibrational energy transfers between the fast and slow time scales were observed among the large-amplitude collective coordinates while the other small-amplitude motions are regarded as thermal noise. Analysis employing a Gaussian-based measure revealed that the time scales of the energy redistribution in the subspace spanned by such large-amplitude collective coordinates are slow compared to the other small-amplitude coordinates. Future prospects of the method are discussed in detail.

Phase transition in dimer liquids

We study the phase transition in a system composed of dimers interacting with each other via a nearest-neighbor (NN) exchange J and competing interactions taken from a truncated dipolar coupling. Each dimer occupies a link between two nearest sites of a simple cubic lattice. We suppose that dimers are self-avoiding and can have only three orientations, which coincide with the x, y or z direction. The interaction J is attractive if the two dimers are parallel to each other at the NN distance, zero otherwise. The truncated dipolar interaction is characterized by two parameters: its amplitude D and the cutoff distance rc. Using the steepest descent method, we determine the ground-state (GS) configuration as functions of D and rc. We then use Monte Carlo simulations to investigate the nature of the low-temperature phase and to determine characteristics of the phase transition from the ordered phase to the disordered phase at high temperatures at a given dimer concentration. We show that as the temperature increases, dimers remain in the compact state and the transition from the low-T compact phase to the disordered phase where dimers occupy the whole space is of second order when D is small, but becomes of first order for large enough D, for both polarized and nonpolarized dimers. This transition has a resemblance to the unfolding polymer transition. The effect of rc is discussed.

Dynamic Solvation and Coupling of the Hydration Shell of ZnII-Substituted Cytochrome c in the Presence of Guanidinium Ions

The fluorescence Stokes shift (FSS) response of Zn(II)-substituted cytochrome c (ZnCytc) is transformed from a monotonic red-shifting response in water to a bidirectional response with much slower time constants in the presence of low concentrations of guanidinium (Gdm(+)) ions. The FSS response in water observed over the 100 ps to 10 ns range has two exponential components with time constants of 135 ps and 1.6 ns that account for a total shift of 30 cm(-1), about one-half of the solvation reorganization energy. In contrast, in the presence of only 0.25 M Gdm(+), the FSS response initially shifts 21 cm(-1) to the blue with a 820 ps time constant and then shifts 60 cm(-1) back to the red with a 3.5 ns time constant. The effect of Gdm(+) on the FSS response effectively saturates at 1.0 M, well below the 1.75 M midpoint of the two-state unfolding transition. These results establish that the FSS response in ZnCytc includes a significant contribution from the surrounding hydration shell, which assumes a perturbed hydrogen-bonding network owing to the binding of Gdm(+) ions to the protein surface. The blue-shifting part of the FSS response arises from a light-induced conformational change that expands the protein- and solvent-derived cavity around the excited-state Zn(II) porphyrin. This non-polar part of the solvation response is enhanced in the presence of Gdm(+) because the protein/solvent surroundings of the Zn(II) porphyrin are effectively more flexible than in water. The enhanced flexibility in the presence of Gdm(+) increases the amplitudes and accordingly lengthens the correlation time scales for the protein and hydration-shell fluctuations that contribute to the FSS response.

Fluorescence Correlation Spectroscopy Study on the Effects of the Shape and Size of a Protein on Its Diffusion Inside a Crowded Environment

Fluorescence correlation spectroscopy (FCS) has been commonly used to study the diffusional and conformational fluctuations of labeled molecules at single-molecule resolution. Here, we explored the applications of FCS inside a polyacrylamide gel to study the effects of molecular weight and molecular shape in a crowded environment. To understand the effect of molecular weight, we carried out FCS experiments with four model systems of different molecular weights in the presence of varying concentrations of acrylamide. The correlation curves were fit adequately using a model containing two diffusing components: one representing unhindered diffusion and one representing slow hindered diffusion in the gel phase. A large number of measurements carried out at different randomly chosen spots on a gel were used to determine the most probable diffusion time values using Gaussian distribution analysis. The variation of the diffusivity with the molecular weight of the model systems could be represented well using the effective medium model. This model assumes a combination of hydrodynamic and steric effects on solute diffusivity. To study the effects of solute shape, FCS experiments were carried inside a urea gradient gel to probe the urea-induced unfolding transition of Alexa488Maleimide-labeled bovine serum albumin. We showed that the scaling behavior, relating the hydrodynamic radius and the number of amino acids, changes inside an acrylamide gel for both folded and unfolded proteins. We showed further that crowding induced by a polyacrylamide gel increases the resolution of measuring the difference in hydrodynamic radii between the unfolded and folded states.

Structural and functional characterization of the MERIT40 to understand its role in DNA repair

MERIT40 (MEdiator of RAP80 Interaction and Targeting 40) is a novel associate of the BRCA1-complex and plays an essential role in DNA damage repair. It is the least characterized protein of BRCA1-complex and mainly responsible for maintaining the complex integrity. However, its structural and functional aspects of regulating the complex stability still remain elusive. Here, we carried out a comprehensive examination of MERIT40 biophysical properties and identified its novel interacting partner which would help to understand its role in BRCA1-complex. The recombinant protein was purified by affinity chromatography and unfolding pathway was determined using spectroscopic and calorimetric methods. Molecular model was generated using combinatorial approaches of modeling, and monomer–monomer docking was carried out to identify dimeric interface. Disordered region of MERIT40 was hatchet using trypsin and chymotrypsin to illustrate the existence of stable domain whose function was speculated through DALI search. Our findings suggest that MERIT40 forms a dimer in a concentration-independent manner. Its central region shows remarkable stability towards the protease digestion and has structural similarity with vWA-like region, a domain mainly present in complement activation factors. MERIT40 undergoes a three-state unfolding transition pathway with a dimeric intermediate. It interacts with adaptor molecule of BRCA1-complex, called ABRAXAS, thus help in extending the bridging interaction among various members which further stabilizes the whole complex. The results presented in this paper provide first-hand information on structural and folding behavior of MERIT40. These findings will help in elucidating the role of protein–protein interactions in stabilization of BRCA1-complex.

Large Extent of Disorder in Adenomatous Polyposis Coli Offers a Strategy to Guard Wnt Signalling against Point Mutations

Mutations in the central region of the signalling hub Adenomatous Polyposis Coli (APC) cause colorectal tumourigenesis. The structure of this region remained unknown. Here, we characterise the Mutation Cluster Region in APC (APC-MCR) as intrinsically disordered and propose a model how this structural feature may contribute to regulation of Wnt signalling by phosphorylation. APC-MCR was susceptible to proteolysis, lacked α-helical secondary structure and did not display thermal unfolding transition. It displayed an extended conformation in size exclusion chromatography and was accessible for phosphorylation by CK1ε in vitro. The length of disordered regions in APC increases with species complexity, from C. elegans to H. sapiens. We speculate that the large disordered region harbouring phosphorylation sites could be a successful strategy to stabilise tight regulation of Wnt signalling against single missense mutations.