Unfolding Kinetics Research Articles

Effective protocol for the investigation of physicochemical and conformational stability and aggregation kinetics measurements of therapeutic IgG2 monoclonal antibody

Characterisation of monoclonal antibodies (mAbs) represents an ongoing challenge due to their diverse 3-dimensional structures that can affect their stability, immunogenicity and/or toxicity. Although circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure in solutions, there is a pressing need for an improvement in current practices in applying the technique for batch QC. There is a lack of experimental evidence in the literature which is concerned with improving the current practices. This work is based on an effective protocol for the study of IgG2a stability in solution using the simultaneous measurements of absorbance, turbidity and CD. A novel approach has been developed for the study of the effects of pH and additives with minimum protein shock that may cause premature aggregation and deceptive results. A computer programme has been designed for the rapid and simultaneous analysis of aggregation during UV and CD measurements, also, highlighting instrumental variations. Temperature stability determination, elucidation of unfolding pathways and aggregation kinetics were estimated with accuracy. This experimental approach provides important information about mAbs physicochemical and conformational stability, helps distinguish between unfolded, refolded, aggregated, and flocculated states and is an excellent tool in the development of therapeutic antibodies.

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na+/K+-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na+ form of Tel23 G4, which is the same hybrid form as the K+ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na+ form to K+ form during Na+/K+ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na+/K+ spectral conversion. In addition, the K+ titration monitored by NMR reveals that the Na+/K+ exchange in Tel23 G4 is a two-step process. The addition of K+ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na+/K+ exchange and insight into the mechanism of Na+/K+ structural conversion in human telomeric G4s.

Unfolding Kinetics of Human Telomeric G-Quadruplexes Studied by NMR Spectroscopy

Characterization of the unfolding kinetics of G-quadruplexes (G4s) is the key to a better understanding of the biological function of G4s and is important for biomedical research and material design. Of interest is that slight variations of human telomeric sequences can form different types of G4 structures. In general, there is a correlation between unfolding kinetics and thermal stability. Here we examined this correlation by first systematic analysis of the unfolding kinetics of a variety of telomeric G4 structures using the real-time imino proton NMR spectra of DNA hybridization and hydrogen-deuterium exchange (HDX). We then measured the melting temperature (Tm) and determined the Gibbs free energy (ΔG) of these G4 structures using differential scanning calorimetry (DSC). Our results showed that both Tm and ΔG are slightly structure-dependent, except the Tm of the parallel G4 structure is ∼10 °C higher than that of nonparallel G4 structures. The hybridization results showed that the decay times of different imino proton signals for each telomeric G4 structure are quite similar, which are also consistent with the time constant of the central G-tetrad obtained from HDX measurements. It is suggested that global unfolding is the rate-determining step for HDX, and each real-time imino proton NMR measurement can provide the intrinsic unfolding rate constant. The key finding is that the unfolding times of these various G4 structures are quite different and show no correlation between thermal stability and unfolding kinetics. Our results raised an issue that the folding and unfolding kinetics is more relevant for better understanding of biological function of G4 structures.

Dual Focus Fluorescence Cross-Correlation Spectroscopy for the Investigation of Biomolecule Folding and Binding in Flowing Liquids

Fluorescence correlation spectroscopy measures the time constants for rate processes giving rise to fluorescence intensity fluctuations observed from one or more microscopic sub-volumes of a nanomolar aqueous solution containing the biomolecules of interest. In our approach, the analyte molecules flow through an electrophoresis capillary under the influence of pressure driven flow or electrophoretic flow and are probed by two spatially offset probe volumes in such a way that the molecules flow sequentially from one probe volume to the next. Fluorescence fluctuations are analyzed from each probe volume independently using autocorrelation analysis, and from the two spatially offset probe volumes using cross-correlation analysis. In this way, we are able to resolve fluorescence fluctuation time constants arising from diffusion, flow, triplet blinking, and conformational fluctuations. Conformational fluctuations are monitored by quenching and unquenching of dye-quencher molecules attached to DNA or RNA hairpin structures and probe the folding and unfolding kinetics of the hairpins. In this presentation, we will discuss recent results that show how base stacking within the loop region of the DNA and RNA hairpins alters the kinetics and thermodynamic stability of the hairpins. We will also discuss binding and unbinding of counterions to individual nucleotides as they flow through the capillary under the influence of an applied electric field. Emphasis will be placed on how the desired information can be extracted using our unique approach to fluorescence correlation spectroscopy.

Osmolyte Effects on the Unfolding Pathway of β-Lactoglobulin

There are large amounts of osmolytes inside cells, which impact many physiological processes by complicated mechanisms. The osmolyte effects on the stability and folding of proteins have been studied in detail using simple two-state folding proteins. However, many important functional proteins fold in complex pathways involving various intermediates. Little is known about the osmolyte effects on the folding and unfolding of these proteins. It is noted that β-lactoglobulin (BLG) is an example of such proteins, whose unfolding involves an obvious intermediate state. Using equilibrium chemical denaturation and stopped-flow kinetics, we investigate the unfolding of BLG in the presence of different osmolytes, e.g., glycerol, ethylene glycol (EG) and poly(ethylene glycol)400 (PEG400). It is found that all these osmolytes can stabilize the unfolding intermediate by modulating the relative unfolding kinetics of the native and the intermediate states. The stabilization effects are similar for EG and PEG400 but distinct for glycerol. Since the unfolding intermediates of many proteins are directly related to protein misfolding diseases, evaluation of the osmolyte effects for the unfolding of these proteins in vitro should be beneficial for the understanding of the occurrence of the related diseases in vivo.

The Utilization of Competing Unfolding Pathways of Monellin Is Dictated by Enthalpic Barriers

Direct evidence of the presence of competing pathways for the folding or unfolding reactions of proteins is difficult to obtain. A direct signature for multiple pathways, seen so far only rarely in folding or unfolding studies, is an upward curvature in the dependence of the logarithm of the observed rate constant (λU) of folding on denaturant concentration. In this study, the unfolding mechanism of the wild-type (wt) and E24A variants of monellin has been investigated, and both variants are shown to display upward curvatures in plots of log λU versus denaturant concentration. Curvature is distinctly more pronounced for E24A than for the wt protein. Kinetic unfolding studies of E24A were conducted over a range of denaturant concentrations and across a range of temperatures, and the kinetic data were globally analyzed assuming two parallel pathways L and H, which proceed through transition states TS(L) and TS(H), respectively. The observation of the upward curvature in the unfolding kinetics permitted a thermodynamic analysis of how unfolding switches from one pathway to the other upon a change in unfolding conditions. The m −N and ΔCp values indicate that TSL is more compact than TS(H). The major contribution to the free energy of activation on either pathway is seen to be enthalpic and not entropic in origin.

The interplay between protein stability and dynamics in conformational diseases: The case of hPGK1 deficiency

Conformational diseases often show defective protein folding efficiency in vivo upon mutation, affecting protein properties such as thermodynamic stability and folding/unfolding/misfolding kinetics as well as the interactions of the protein with the protein homeostasis network. Human phosphoglycerate kinase 1 (hPGK1) deficiency is a rare inherited disease caused by mutations in hPGK1 that lead to loss-of-function. This disease offers an excellent opportunity to explore the complex relationships between protein stability and dynamics because of the different unfolding mechanisms displayed towards chemical and thermal denaturation. This work explores these relationships using two thermostable mutants (p.E252A and p.T378P) causing hPGK1 deficiency and WT hPGK1 using proteolysis and chemical denaturation. p.T378P is degraded ~30-fold faster at low protease concentrations (here, the proteolysis step is rate-limiting) and ~3-fold faster at high protease concentrations (where unfolding kinetics is rate-limiting) than WT and p.E252A, indicating that p.T378P is thermodynamically and kinetically destabilized. Urea denaturation studies support the decrease in thermodynamic stability and folding cooperativity for p.T378P, as well as changes in folding/unfolding kinetics. The present study reveals changes in the folding landscape of hPGK1 upon mutation that may affect protein folding efficiency and stability in vivo, also suggesting that native state stabilizers and protein homeostasis modulators may help to correct folding defects in hPGK1 deficiency. Moreover, detailed kinetic proteolysis studies are shown to be powerful and simple tools to provide deep insight into mutational effects on protein folding and stability in conformational diseases.

Testing the diffusing boundary model for the helix–coil transition in peptides

The dynamics of peptide α-helices have been studied extensively for many years, and the kinetic mechanism of the helix-coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix-coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7⋅10(7) (amino acids)(2)/s or 6.1⋅10(-9) cm(2)/s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix-coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a ϕf-value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding (ϕf = 0) in agreement with a diffusing boundary mechanism.

Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.

Using D-amino acids to delineate the mechanism of protein folding: Application to Trp-cage

Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding–unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and d-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.

Kinetic and thermodynamic properties of pseudomonas fluorescence lipase upon addition of proline

The effect of proline on refolding and unfolding kinetics as well as activity of lipase from Pseudomonas fluorescens was determined using stopped-flow fluorescence and UV/Vis absorbance spectroscopy. Enzyme assay at different concentrations of proline revealed that activity of enzyme reaches maximum at 0.6M concentration of proline. Kinetic measurements showed that refolding rate is considerably accelerated in the presence of 0.6M proline. Unfolding kinetic traces were fitted to double exponential function, and it was revealed that lipase molecules were unfolded via two different pathways. Fast unfolding rate constant decreased, while slow one did not change significantly upon addition of proline.

Identity of Hinge Residues Defines Stability and Kinetics of Spectrin Tetramer Interaction

Spectrin is an elongated structural protein which forms, through inter-subunit interaction, a two-dimensional lattice-work of support for the cytoskeleton and membrane of a cell. The head-to-head interaction of the α- and β subunits, the “tetramer interaction,” is of particular interest, as here two partially unstructured domains undergo a coupled binding and folding reaction to yield a new three-helix bundled domain. In mammals, spectrin tetramers are found in two isoforms, SpI and SpII. SpI, the erythrocyte spectrin, forms relatively weak tetramers that exhibit slow folding and unfolding kinetics, believed to contribute to the intrinsic flexibility and durability of the red blood cell. In contrast SpII, the non-erythrocyte spectrin, rapidly associates to forms tetramers three orders of magnitude more stable than SpI. Intriguingly, the choice of β-subunit isoform does not dramatically alter the properties of the tetramer, indicating that difference in α-subunit isoform confers the majority of increased stability and association rate. Previous work has attributed this effect to a single residue (G46 in SpIα versus R37 in SpIIα) linking the N-terminal partially structured tail to the fully folded first domain of the α-subunit. In order to investigate the importance of helical propensity and charge character to the stability and association kinetics of the spectrin tetramer, we have made a series of point mutants in SpIα and SpIIα. Our early data suggest that increasing the helical propensity of residue 46 in SpIα by mutation to alanine accelerates the association kinetics, but does not alter the thermodynamic stability of the complex, while mutation to arginine dramatically increases the stability and folding kinetics.

A Comparative Study of Mechanical Stability of Circular Permutants during Co-Translocational Unfolding of Proteins through Mitochondrial Pore

Mitochondrial import machinery catalyses unfolding of the native precursor proteins by trapping some faster local unfolding fluctuations due to specific secondary structural element adjacent to the targeting sequence. On the rupture of the first resistant structure, the rest of the protein unfolds rapidly by cooperative unfolding during import into the mitochondrial matrix. The process of circular permutation on the protein chain can give important information about the connectivity, structure and folding or unfolding kinetics which guides its translocation and subsequent function in the cell. To study the process of co-translocational unfolding and its dependence on the secondary structure at the N-terminus of the protein imported, we conduct a comparative molecular dynamics study of circular permutants of Dihydrofolate reductase(DHFR) using atomistic model in CHARMM. Six Circular Permutants - CP25, CP38, CP78, CP97, CP108, CP133 - are generated such that the new N-terminus leads either to an alpha helix or a beta-strand. using Steered Molecular Dynamics we compute the work distributions for the forced unfolding of each of the CP's and native DHFR using two processes - unfolding through the geometrical constriction of the model pore as in mitochondrial translocation and mechanical unfolding with the C-terminus fixed. In both cases the unfolding force is applied at the N-terminus. A comparison of the free energy profile along the reaction coordinate for each circular permutant can lead to identification of different unfolding pathways and hence import efficiency based on first resistant structure adjacent to the targeting sequence.

Study of pH-Induced Folding and Unfolding Kinetics of the DNA i-Motif by Stopped-Flow Circular Dichroism

Using the stopped-flow circular dichroism (SFCD) technique, we investigate the kinetics of the pH-induced folding and unfolding process of the DNA i-motif. The results show that the molecule can fold or unfold on a time scale of 100 ms when the solution pH is changed. It is also found that the folding and unfolding rates strongly depend on the solution pH. On the basis of quantitative data, we propose theoretical models to decipher the folding and unfolding kinetics. Our models suggest that the cooperativity of protons is crucial for both the folding and unfolding process. In the unfolding process, the cooperative neutralization of two protons (out of the total six protons in the i-motif molecule) is the only rate-limiting step. In the folding process, there exists a critical step in which three protons bind cooperatively to the DNA strand. These results offer an in-depth understanding of the folding and unfolding kinetics of the DNA i-motif and may give precise guidance for constructing novel nanodevices based on the DNA i-motif.

Force-Clamp Analysis Techniques Give Highest Rank to Stretched Exponential Unfolding Kinetics in Ubiquitin

Force-clamp spectroscopy reveals the unfolding and disulfide bond rupture times of single protein molecules as a function of the stretching force, point mutations, and solvent conditions. The statistics of these times reveal whether the protein domains are independent of one another, the mechanical hierarchy in the polyprotein chain, and the functional form of the probability distribution from which they originate. It is therefore important to use robust statistical tests to decipher the correct theoretical model underlying the process. Here, we develop multiple techniques to compare the well-established experimental data set on ubiquitin with existing theoretical models as a case study. We show that robustness against filtering, agreement with a maximum likelihood function that takes into account experimental artifacts, the Kuiper statistic test, and alignment with synthetic data all identify the Weibull or stretched exponential distribution as the best fitting model. Our results are inconsistent with recently proposed models of Gaussian disorder in the energy landscape or noise in the applied force as explanations for the observed nonexponential kinetics. Because the physical model in the fit affects the characteristic unfolding time, these results have important implications on our understanding of the biological function of proteins.

Real-Time Protein Unfolding: A Method for Determining the Kinetics of Native Protein Denaturation using a Quantitative Real-Time Thermocycler

Protein stability can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. Here we present a new protocol to analyze protein unfolding kinetics using a quantified real-time thermocycler. This technique enables the analysis of a wide range of denaturants (and their interactions with temperature change) on protein stability in a multi-well platform, where samples can be run in parallel under virtually identical conditions and with highly sensitive detection. Using this set-up, researchers can evaluate the half-maximal rate of protein denaturation (K(nd)), maximum rate of denaturation (D(max)), and the cooperativity of individual denaturants in protein unfolding (µ-coefficient). Both lysozyme and hexokinase are used as model proteins and urea as a model denaturant to illustrate this new method and the kinetics of protein unfolding that it provides. Overall, this method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay, providing estimates of denaturation kinetics that have been previously inaccessible.

Programmed −1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding

Programmed -1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.

Single-Molecule Stochastic Resonance

Stochastic resonance (SR) is a well-known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic (random or probabilistic) noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively transitions from folded to unfolded configurations under the action of an oscillating mechanical force applied with optical tweezers. By varying the frequency of the force oscillation, we investigate the folding and unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measure several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that a good quantifier of the SR is the signal-to-noise ratio (SNR) of the spectral density of measured fluctuations in molecular extension of the DNA hairpins. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance-matching condition. Finally, we carry out experiments on short hairpins that show how SR might be useful for enhancing the detection of conformational molecular transitions of low SNR.Received 14 March 2012DOI:https://doi.org/10.1103/PhysRevX.2.031012This article is available under the terms of the Creative Commons Attribution 3.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.Published by the American Physical Society

Sequential four‐state folding/unfolding of goat α‐lactalbumin and its N‐terminal variants

Equilibria and kinetics of folding/unfolding of α-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped-flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst-phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding-limb curvature was interpreted in terms of accumulation of the burst-phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding-limb curvature. We thus assumed a sequential four-state mechanism, in which the folding from the burst-phase intermediate takes place via two transition states separated by a high-energy intermediate. We estimated changes in the free energies of the burst-phase intermediate and two transition states, caused by the N-terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca(2+)-binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo-wild-type α-lactalbumin in future studies.

Dynamic Prestress in a Globular Protein

A protein at equilibrium is commonly thought of as a fully relaxed structure, with the intra-molecular interactions showing fluctuations around their energy minimum. In contrast, here we find direct evidence for a protein as a molecular tensegrity structure, comprising a balance of tensed and compressed interactions, a concept that has been put forward for macroscopic structures. We quantified the distribution of inter-residue prestress in ubiquitin and immunoglobulin from all-atom molecular dynamics simulations. The network of highly fluctuating yet significant inter-residue forces in proteins is a consequence of the intrinsic frustration of a protein when sampling its rugged energy landscape. In beta sheets, this balance of forces is found to compress the intra-strand hydrogen bonds. We estimate that the observed magnitude of this pre-compression is enough to induce significant changes in the hydrogen bond lifetimes; thus, prestress, which can be as high as a few 100 pN, can be considered a key factor in determining the unfolding kinetics and pathway of proteins under force. Strong pre-tension in certain salt bridges on the other hand is connected to the thermodynamic stability of ubiquitin. Effective force profiles between some side-chains reveal the signature of multiple, distinct conformational states, and such static disorder could be one factor explaining the growing body of experiments revealing non-exponential unfolding kinetics of proteins. The design of prestress distributions in engineering proteins promises to be a new tool for tailoring the mechanical properties of made-to-order nanomaterials.