Unfolding Kinetics Research Articles

Metformin-Induced Invertase Unfolding: Enzyme Kinetics and Activity Regulation.

The effects of metformin on invertase activity and its inhibition on sucrose digestion were studied. The rapid unfolding kinetics of invertases, followed a two-state model with an inactive intermediate formation. The dynamic interaction between metformin and invertase caused the secondary structure of the enzyme to become less β-sheet, more α-helix, and random coiling oriented, which weakened the binding force between enzyme and its substrate. Metformin acted as a chaotrope and disrupted the hydrogen bonds of water, which facilitated the unfolding of invertase. However, some sugar alcohols, which promoted the H-bond formation of water, could repair the secondary structure of metformin-denatured invertase and therefore regulate the enzyme activity. This research enriches our understanding of the mechanism of enzyme unfolding induced by guanidine compounds. Moreover, because metformin and sugar substitutes are of concern to diabetes, this research also provides useful information for understanding the activity of the digestive enzyme that coexists with metformin and sugar alcohols.

Ultrasonic Sensor: A Fast and Non-Destructive System to Measure the Viscosity and Density of Molecular Fluids.

This study presents the design and development of an ultrasonic sensor as a fundamental tool for characterizing the properties of fluids and biofluids. The analysis primarily focuses on measuring the electrical parameters of the system, which correlate with the density and viscosity of the solutions, in sample volumes of microliters and with high temporal resolution (up to 1 data point per second). The use of this sensor allows the fast and non-destructive evaluation of the viscosity and density of fluids deposited on its free surface. The measurements are based on obtaining the impedance versus frequency curve and the phase difference curve (between current and voltage) versus frequency. In this way, characteristic parameters of the transducer, such as the resonance frequency, phase, minimum impedance, and the quality factor of the resonant system, can characterize variations in density and viscosity in the fluid under study. The results obtained revealed the sensor's ability to identify two parameters sensitive to viscosity and two parameters sensitive to density. As a proof of concept, the unfolding of the bovine albumin protein was studied, resulting in a curve that reflects its unfolding kinetics in the presence of urea.

Structurally different chemical chaperones show similar mechanical roles with independent molecular mechanisms.

Osmolytes are well known to protect the protein structure against different chemical and physical denaturants. Since their actions with protein surfaces are mechanistically complicated and context dependent, the underlying molecular mechanism is not fully understood. Here, we combined single-molecule magnetic tweezers and molecular dynamics (MD) simulation to explore the mechanical role of osmolytes from two different classes, trimethylamine N-oxide (TMAO) and trehalose, as mechanical stabilizers of protein structure. We observed that these osmolytes increase the protein L mechanical stability by decreasing unfolding kinetics while accelerating the refolding kinetics under force, eventually shifting the energy landscape toward the folded state. These osmolytes mechanically stabilize the protein L and plausibly guide them to more thermodynamically robust states. Finally, we observed that osmolyte-modulated protein folding increases mechanical work output up to twofold, allowing the protein to fold under a higher force regime and providing a significant implication for folding-induced structural stability in proteins.

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs.

Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity.

Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two β-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Single-Molecule Optical Tweezers As a Tool for Delineating the Mechanisms of Protein-Processing Mechanoenzymes.

Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA unwinding to protein degradation. Protein-processing mechanoenzymes either remodel the protein structures or translocate them across cellular compartments in an energy-dependent manner. Optical-tweezer-based single-molecule force spectroscopy assays have divulged information on details of chemo-mechanical coupling, directed motion, as well as mechanical forces these enzymes are capable of generating. In this review, we introduce the working principles of optical tweezers as a single-molecule force spectroscopy tool and assays developed to decipher the properties such as unfolding kinetics, translocation velocities, and step sizes by protein remodeling mechanoenzymes. We focus on molecular motors involved in protein degradation and disaggregation, i.e., ClpXP, ClpAP, and ClpB, and insights provided by single-molecule assays on kinetics and stepping dynamics during protein unfolding and translocation. Cellular activities such as protein synthesis, folding, and translocation across membranes are also energy dependent, and the recent single-molecule studies decoding the role of mechanical forces on these processes have been discussed.

Droplet-Based Microfluidic Temperature-Jump Platform for the Rapid Assessment of Biomolecular Kinetics.

Protein folding, unfolding, and aggregation are important in a variety of biological processes and intimately linked to "protein misfolding diseases". The ability to perform experiments at different temperatures allows the extraction of important information regarding the kinetics and thermodynamics of such processes. Unfortunately, conventional stopped-flow methods are difficult to implement, generate limited information, and involve complex sample handling. To address this issue, we present a temperature-controlled droplet-based microfluidic platform that allows measurement of reaction kinetics on millisecond to second timescales and at temperatures between ambient and 90 °C. The utility of the microfluidic platform for measuring fast biomolecular kinetics at high temperatures is showcased through the investigation of the unfolding kinetics of haloalkane dehalogenases and the elongation of fibrils composed of the amyloid β peptide associated with Alzheimer's disease. In addition, a deep-ultraviolet (UV) fluorescence microscope was developed for the on-chip recording of protein intrinsic fluorescence spectrum originating from aromatic amino acid residues. We envision that the developed optofluidic platform will find wide applicability in the analysis of biological processes, such as protein refolding and phase separation.

Mechanical regulation of talin through binding and history-dependent unfolding.

Talin is a force-sensing multidomain protein and a major player in cellular mechanotransduction. Here, we use single-molecule magnetic tweezers to investigate the mechanical response of the R8 rod domain of talin. We find that under various force cycles, the R8 domain of talin can display a memory-dependent behavior: At the same low force (<10 pN), the same protein molecule shows vastly different unfolding kinetics. This history-dependent behavior indicates the evolution of a unique force-induced native state. We measure through mechanical unfolding that talin R8 domain binds one of its ligands, DLC1, with much higher affinity than previously reported. This strong interaction can explain the antitumor response of DLC1 by regulating inside-out activation of integrins. Together, our results paint a complex picture for the mechanical unfolding of talin in the physiological range and a new mechanism of function of DLC1 to regulate inside-out activation of integrins.

Label‐Free Detection and Translocation Dynamics Study of Single‐Molecule Herceptin Using Solid‐State Nanopores

AbstractHerceptin (or trastuzumab) is an important therapeutic monoclonal antibody (mAb) used in the treatment of HER2‐positive breast cancer. Real‐time counting and characterization of Herceptin is a fundamental step in the field of disease‐related diagnosis and therapy. Solid‐state nanopore‐based biosensors have been proved to hold great potential in characterizing the properties of proteins at the single‐molecule level for in vitro diagnosis. Here, the label‐free detection and detailed translocation dynamics study of Herceptin using solid‐state nanopores are demonstrated. By constricting nanopore size close to the size of Herceptin, the detection sensitivity and temporal resolution have been significantly improved, allowing the delicate probing of the structural information of single‐molecule Herceptin. Therefore, three types of Herceptin translocation events are identified through nanopores, single‐level, multi‐level and spike‐like events, emerged at different voltages regimes, indicating the unfolding kinetics of Herceptin under electric field. The potential influence of a high electric field on complex biomolecules is highlighted and a novel prospective platform is provided for label‐free detection of single‐molecule therapeutic monoclonal antibodies via solid‐state nanopores as a miniaturized biomedical device.

L-Arginine inhibits the activity of α-amylase: Rapid kinetics, interaction and functional implications

In this work, the rapid unfolding kinetics of pancreas α-amylase (PPA) induced by l-arginine and the interaction mechanism were investigated. The unfolding followed a first-level reaction kinetics equation, without intermediates. l-arginine interacted with PPA though diffusion-controlled process rather than complexion. The interaction between l-arginine and PPA resulted in a pronounced decrease in β-sheet and a significant increase in random coil, and thereby the enzyme activity decreased. However, the unfolding of PPA could be compensated and the second structure change could be recovered to some extent by the macromolecular crowded medium of Pluronics. Further insight into the mechanism disclosed that the broken H-bond network of water may contribute to PPA unfolding. This work provides a new perspective on the interaction of l-arginine with digestive enzyme. The unfolding mechanism of enzymes by may help to understand the effects of other structurally similar drugs, which is of concern in food-drug interactions.

Nonexponential kinetics captured in sequential unfolding of polyproteins over a range of loads.

While performing under mechanical loads in vivo, polyproteins are vitally involved in cellular mechanisms such as regulation of tissue elasticity and mechano-transduction by unfolding their comprising domains and extending them. It is widely thought that the process of sequential unfolding of polyproteins follows an exponential kinetics as the individual unfolding events exhibit identical and identically distributed (iid) Poisson behavior. However, it was shown that under high loads, the sequential unfolding kinetics displays nonexponential kinetics that alludes to aging by a subdiffusion process. Statistical order analysis of this kinetics indicated that the individual unfolding events are not iid, and cannot be defined as a Poisson (memoryless) process. Based on numerical simulations it was argued that this behavior becomes less pronounced with lowering the load, therefore it is to be expected that polyproteins unfolding under lower forces will follow a Poisson behavior. This expectation serves as the motivation of the current study, in which we investigate the effect of force lowering on the unfolding kinetics of Poly-L8 under varying loads, specifically high (150, 100 ​pN) and moderate-low (45, 30, 20 ​pN) forces. We found that a hierarchy among the unfolding events still exists even under low loads, again resulting in nonexponential behavior. We observe that analyzing the dwell-time distributions with stretched-exponentials and power laws give rise to different phenomenological trends. Using statistical order analysis, we demonstrated that even under the lowest load, the sequential unfolding cannot be considered as iid, in accord with the power law distribution. Additional free energy analysis revealed the contribution of the unfolded segments elasticity that scales with the force on the overall one-dimensional contour of the energy landscape, but more importantly, it discloses the hierarchy within the activation barriers during sequential unfolding that account for the observed nonexponentiality.

High-resolution MD Simulation Studies to Get Mechanistic Insights into the Urea-induced Denaturation of Human Sphingosine Kinase 1.

Sphingosine kinase 1 (SPhK1) is a crucial signaling enzyme involved in cell proliferation, cellular survival, stimulation of angiogenesis, and apoptosis prevention. Recently, we have reported the unfolding kinetics of SPhK1 using molecular dynamics (MD) simulation, circular dichroism, and fluorescence spectroscopy. We found that SPhK1 showed a biphasic unfolding with an intermediate state (~ 4.0 M urea). We aim to understand the impact of MD simulation duration on the structure, function, and dynamics of proteins. In order to get deeper insights into the folding mechanism, an extended MD simulation is required. Here, we extended the MD simulations time scale from 100 to 300 ns on SPhK1 at increasing urea concentration to explore structural changes in the SPhK1. The results suggested a constant form of the unfolding of SPhK1 upon extending the simulation time scale at different urea concentrations. Furthermore, we showed step by step unfolding and percentage of secondary structure contents in SPhK1 under the influence of urea at each concentration. The results from the current work revealed a uniform pattern of the SPhK1 unfolding at different urea concentrations. This study provides deeper mechanistic insights into the urea-induced denaturation of SPhK1.

Molecular understanding of calorimetric protein unfolding experiments

Testing and predicting protein stability gained importance because proteins, including antibodies, became pharmacologically relevant in viral and cancer therapies. Isothermal scanning calorimetry is the principle method to study protein stability. Here, we use the excellent experimental heat capacity Cp(T) data from the literature for a critical inspection of protein unfolding as well as for the test of a new cooperative model. In the relevant literature, experimental temperature profiles of enthalpy, Hcal(T), entropy, Scal(T), and free energy, Gcal(T) are missing. First, we therefore calculate the experimental Hcal(T), Scal(T), and Gcal(T) from published Cp(T) thermograms. Considering only the unfolding transition proper, the heat capacity and all thermodynamic functions are zero in the region of the native protein. In particular, the free energy of the folded proteins is also zero and Gcal(T) displays a trapezoidal temperature profile when cold denaturation is included. Second, we simulate the DSC-measured thermodynamic properties with a new molecular model based on statistical-mechanical thermodynamics. The model quantifies the protein cooperativity and predicts the aggregate thermodynamic variables of the system with molecular parameters only. The new model provides a perfect simulation of all thermodynamic properties, including the observed trapezoidal Gcal(T) temperature profile. Importantly, the new cooperative model can be applied to a broad range of protein sizes, including antibodies. It predicts not only heat and cold denaturation but also provides estimates of the unfolding kinetics and allows a comparison with molecular dynamics calculations and quasielastic neutron scattering experiments.

Single-Molecule Force Spectroscopy Studies of Missense Titin Mutations That Are Likely Causing Cardiomyopathy.

The giant muscle protein titin plays important roles in heart function. Mutations in titin have emerged as a major cause of familial cardiomyopathy. Missense mutations have been identified in cardiomyopathy patients; however, it is challenging to distinguish disease-causing mutations from benign ones. Given the importance of titin mechanics in heart function, it is critically important to elucidate the mechano-phenotypes of cardiomyopathy-causing mutations found in the elastic I-band part of cardiac titin. Using single-molecule atomic force microscopy (AFM) and equilibrium chemical denaturation, we investigated the mechanical and thermodynamic effects of two missense mutations, R57C-I94 and S22P-I84, found in the elastic I-band part of cardiac titin that were predicted to be likely causing cardiomyopathy by bioinformatics analysis. Our AFM results showed that mutation R57C had a significant destabilization effect on the I94 module. R57C reduced the mechanical unfolding force of I94 by ∼30–40 pN, accelerated the unfolding kinetics, and decelerated the folding. These effects collectively increased the unfolding propensity of I94, likely resulting in altered titin elasticity. In comparison, S22P led to only modest destabilization of I84, with a decrease in unfolding force by ∼10 pN. It is unlikely that such a modest destabilization would lead to a change in titin elasticity. These results will serve as the first step toward elucidating mechano-phenotypes of cardiomyopathy-causing mutations in the elastic I-band.

Single-molecule mechanical folding and unfolding kinetics of armless mitochondrial tRNAArg from Romanomermis culicivorax * *Project supported by the Natural Science Foundation of Guangdong Province, China (Grant No. 2017A030310085) and the Science and Technology Planning Project of Guangdong Province, China (Grant No. 2018A050506034).

The mechanical stability of tRNAs contributes to their biological activities. The mitochondrial tRNAArg from Romanomermis culicivorax is the shortest tRNA ever known. This tRNA lacks D- and T-arms, represents a stem–bulge–stem architecture but still folds into a stable tertiary structure. Although its structure had been reported, studies on its mechanical folding and unfolding kinetic characteristics are lacking. Here, we directly measured the single-molecule mechanical folding and unfolding kinetics of the armless mt tRNAArg by using optical tweezers in different solution conditions. We revealed a two-step reversible unfolding pathway: the first and large transition corresponds to the unfolding of acceptor stem and bulge below 11 pN, and the second and small transition corresponds to the unfolding of anticodon arm at 12 pN–14 pN. Moreover, the free energy landscapes of the unfolding pathways were reconstructed. We also demonstrated that amino acid-chelated Mg2+(aaCM), which mimics the intracellular solution condition, stabilizes the bulge of mitochondrial tRNAArg possibly by reducing the topological constraints or stabilizing the possible local non-canonical base pairings within the bulge region. Our study revealed the solution-dependent mechanical stability of an armless mt tRNA, which may shed light on future mt tRNA studies.

Rapid unfolding of pig pancreas α-amylase: Kinetics, activity and structure evolution

α-Amylase plays an important role in food processing and in-vivo digestion. Many biological functions of α-amylase are affected by unfolding. The pre-steady-state rapid unfolding kinetics of α-amylase remains unknown. In this study, the rapid unfolding kinetics of porcine pancreatic α-amylase (PPA) with guanidine hydrochloride (GdmHCl) were investigated by stopped-flow spectroscopy. Structural characterization of PPA by fluorescence spectroscopy, and molecular dynamics simulation showed that the unfolding process of PPA might start from the internal active center, where the β-sheet structure was destroyed, followed by the exposure of hydrophobic amino acid residues. Further research revealed that GdmHCl denaturized PPA not by complexing with PPA.The surrounding H-bond network of water was changed by GdmHCl. This research improves our understanding of the unfolding kinetics of the PPA on the microsecond scale. It also provides the evidence experimentally of the surrounding water contribution to protein denaturization.

Cys-labeling kinetics of membrane protein GlpG: a role for specific SDS binding and micelle changes?

Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MFSDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MFSDS range, along with exchange from the native state at low MFSDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MFSDS at 0–0.2 MFSDS (i.e., native conditions). Thus, an increase in MFSDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MFSDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.

Non-Markovian modeling of protein folding

We extract the folding free energy landscape and the time-dependent friction function, the two ingredients of the generalized Langevin equation (GLE), from explicit-water molecular dynamics (MD) simulations of the α-helix forming polypeptide [Formula: see text] for a one-dimensional reaction coordinate based on the sum of the native H-bond distances. Folding and unfolding times from numerical integration of the GLE agree accurately with MD results, which demonstrate the robustness of our GLE-based non-Markovian model. In contrast, Markovian models do not accurately describe the peptide kinetics and in particular, cannot reproduce the folding and unfolding kinetics simultaneously, even if a spatially dependent friction profile is used. Analysis of the GLE demonstrates that memory effects in the friction significantly speed up peptide folding and unfolding kinetics, as predicted by the Grote-Hynes theory, and are the cause of anomalous diffusion in configuration space. Our methods are applicable to any reaction coordinate and in principle, also to experimental trajectories from single-molecule experiments. Our results demonstrate that a consistent description of protein-folding dynamics must account for memory friction effects.