Folding Rate Research Articles

Tunable order-disorder continuum in protein-DNA interactions.

DNA-binding protein domains (DBDs) sample diverse conformations in equilibrium facilitating the search and recognition of specific sites on DNA over millions of energetically degenerate competing sites. We hypothesize that DBDs have co-evolved to sense and exploit the strong electric potential from the array of negatively charged phosphate groups on DNA. We test our hypothesis by employing the intrinsically disordered DBD of cytidine repressor (CytR) as a model system. CytR displays a graded increase in structure, stability and folding rate on increasing the osmolarity of the solution that mimics the non-specific screening by DNA phosphates. Electrostatic calculations and an Ising-like statistical mechanical model predict that CytR exhibits features of an electric potential sensor modulating its dimensions and landscape in a unique distance-dependent manner, while DNA plays the role of a non-specific macromolecular chaperone. Accordingly, CytR binds its natural half-site faster than the diffusion-controlled limit and even random DNA conforming to an electrostatic-steering binding mechanism. Our work unravels for the first time the synergistic features of a natural electrostatic potential sensor, a novel binding mechanism driven by electrostatic frustration and disorder, and the role of DNA in promoting distance-dependent protein structural transitions critical for switching between specific and non-specific DNA-binding modes.

Tests of Kramers' Theory at the Single-Molecule Level: Evidence for Folding of an Isolated RNA Tertiary Interaction at the Viscous Speed Limit.

Dissipation and friction influence the conformational dynamics of biological polymers as they traverse barriers on rugged free energy surfaces. It is well established that the "speed limit" for macromolecular folding is dictated by a combination of (i) solvent friction, which depends on solvent viscosity, η, and (ii) internal friction, which is independent of solvent and depends solely on the molecular folding pathway. In this work, single-molecule Förster resonance energy transfer (FRET) confocal spectroscopy is used to study viscosity-dependent folding kinetics of an isolated RNA tertiary motif, that of the GAAA tetraloop receptor, allowing both solvent and internal frictional contributions to be investigated and extracted independently for both flexible PEG- and RNA-based (rU7, rA7) linkers in the unimolecular construct. Specifically, our single-molecule data reveal that (i) folding rate constants scale linearly with the inverse solvent viscosity (η), which supports Kramers'/Grote-Hynes' rate theory for η-dependent RNA folding and that (ii) they provide quantitative upper limits for the intrinsic viscosity, [ηint ≈ 0.1(2) cP], arising from internal friction associated with folding/unfolding of an isolated RNA tertiary interaction. Furthermore, in contrast to strong viscosity-induced shifts in the folding/unfolding rate constants, temperature-dependent studies demonstrate that the enthalpic, entropic, and free energy contributions to the transition state barrier are largely insensitive to the solvent viscosity. This supports a very simple picture for the conformational kinetics of isolated RNA tertiary interactions wherein rate constants for folding/unfolding are both inversely dependent on viscosity and limited by diffusional access to the transition state region on a multidimensional free energy surface. Particularly under cellular conditions, where ηsolv > 1 cp, this suggests that RNAs fold/unfold at a "speed limit" dictated by solvent viscosity and transition-state barrier thermodynamics rather than internal molecular friction.

Effects of Non-native Interactions on Frustrated Proteins Folding under Confinement.

In vitro, kinetically significant non-native interactions have been identified experimentally during the folding of proteins Im7, Im9, and A39V/N53P/V55L Fyn SH3 domain. To understand the role of non-native interactions on the folding of some frustrated proteins in chaperone, we employed native-centric models with and without additional transferable, sequence-dependent non-native hydrophobic interactions to comparatively study the folding behaviors of the three proteins confined in spherical cages. Under purely repulsive confinement, as a decrease of cavity size, the non-native interactions increase, especially in the unfolded state, enhancing the roughness of the folding energy landscape. As a result, the increase in native stability for the three proteins by the model incorporated non-native interactions (db + MJ hϕ model) is much smaller than that by the purely native-centric model (desolvation-barrier (db) model); the acceleration of folding simulated by the db + MJ hϕ model is much slower than that via the db model; in particular, the folding rate of Im7 decreases when reducing the cavity size under zero-denaturant condition. The repulsive confinement can also promote formation of specific non-native contacts in the transition state and favor more folding pathways passing through the misfolded state, leading to a higher population of the misfolded intermediate. In an attractive cage, the attractive interactions could inhibit the formation of intrachain non-native contacts and provide alternate folding pathways to the native state so that the population of the misfolded intermediate decreases when increasing the strength of attractive interaction between the substrate protein and cavity wall. This study should be helpful in general to understand how the chaperonins reshape the folding energy landscape of some frustrated proteins.

Testing Kinetic Identities Involving Transition-Path Properties Using Single-Molecule Folding Trajectories.

Recent advances in single-molecule assays have allowed individual transition paths during the folding of single molecules to be observed directly. We used the transition paths of DNA hairpins having different sequences, measured with high-resolution optical tweezers, to test theoretical relations between the properties of the transition paths and the folding kinetics. We showed that folding and unfolding rates were related to the average transition-path times, as expected from theory, for all hairpins studied. We also found that the probability distribution of transition-path occupancies agreed with the profile of the average velocity along the transition paths for each of the hairpins, as expected theoretically. Finally, we used the latter result to show that the committor probability recovered from the velocity profile matches the committor measured empirically. These results validate the proposed kinetic identities.

Abstract 4841: Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex

Abstract Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target. Citation Format: Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4841.

Non-Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells.

Although biomolecules evolved to function in the cell, most biochemical assays are carried out in vitro. In-cell studies highlight how steric and non-steric interactions modulate protein folding and interactions. VlsE and PGK present two extremes of chemical behavior in the cell: the extracellular protein VlsE is destabilized in eukaryotic cells, whereas the cytoplasmic protein PGK is stabilized. VlsE and PGK are benchmarks in a systematic series of solvation environments to distinguish contributions from non-steric and steric interactions to protein stability, compactness, and folding rate by comparing cell lysate, a crowding agent, ionic buffer and lysate buffer with in-cell results. As anticipated, crowding stabilizes proteins, causes compaction, and can speed folding. Protein flexibility determines its sensitivity to steric interactions or crowding. Non-steric interactions alone predict in-cell stability trends, while crowding provides an offset towards greater stabilization. We suggest that a simple combination of lysis buffer and Ficoll is an effective new in vitro mimic of the intracellular environment on protein folding and stability.

Prediction of kinetics of protein folding with non-redundant contact information.

The majority of the inter-residue distances in a protein structure are correlated given a fixed topology. Here, we investigate whether we are able to predict a structure's folding rate, which is known to depend on the complexity of its fold, while considering only a small, uncorrelated fraction of its contacts. We define an expression for the probabilistic information content associated to the relative position of a pair of amino acid residues in a protein structure. By means of fitting the protein chain to a self-avoiding random walk model, we derive a probability distribution for the distance between residues as a function of their separation along the sequence. We then show that the average information content for all residue pairs in a structure, considered as an estimate of its fold's complexity, is well correlated to the logarithm of its folding rate. Moreover, the same information content measure may be exploited to rank contacts and identify redundancies, allowing the prediction the structure's folding rate with similar accuracy while taking into account less than 5% of its contacts. An implementation of the described model and the experimental data are available at http://github.com/luciano-censoni/sarw-lnkf.

Single-Molecule Mechanical Folding and Unfolding of RNA Hairpins: Effects of Single A-U to A·C Pair Substitutions and Single Proton Binding and Implications for mRNA Structure-Induced -1 Ribosomal Frameshifting.

A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A+·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk -1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, -1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (∼2%). Accordingly, the bottom 2-4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (∼1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes.

Exploring the Influence of Mutation on Transthyretin Aggregation in Heart Disease.

Transthyretin (TTR) is the transporter protein (55 kDa) that carries retinolbinding protein and Thyroxin (T4) in its functional tetramer form. Presence of the mutation in this protein (TTR) may lead to the dissociation of tetramers to monomer which unfolds and self-associates to form amyloid aggregates. Aggregation of this protein has been found to be associated with various lifethreatening disorders such as Coronary Artery Disease (CAD) which is the major cause of mortality and morbidity worldwide. In the present communication, we have predicted mutation prone residues of TTR with the help of suspect server. Substitution (T139R with 95 score) occurring at the thyroid hormone binding site was selected for studying the mutational consequences on TTR. The effect of mutation on stability, functionality, aggregation and folding rate was analyzed by MuPro, DUET, SDM, SNAP2, Polyphen2, PASTA2.0, Aggrescan and Folding RaCe servers. The presence of TTR monomer in CAD plasma has been observed through Western blot analysis. T139R mutation may expose the buried regions of TTR protein which help in the self association and the increase in the stability may help in the TTR deposition. Structural analysis indicated that F and H strands of TTR are more prone to aggregation. Thus, T139R mutation might cause these residues to be aggregation prone and change in folding rate and validated TTR monomer in diseased cases by Western blot analysis. The observed results clearly indicated that the occurrence of this mutation is causing the impact on the structural and functional significance of TTR by interfering in the formation of tetramer. Thus, hindrance created to thyroxin transportation resulted in higher lipid levels in the blood that ultimately might promote the progression of the CAD.

Effect of the geometry of confining media on the stability and folding rate of -helix proteins.

Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D 0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding temperature T f on the size D of a confined medium occurs in larger media for larger proteins is correct only in spherical geometry, whereas the opposite is true in the two other geometries that we study. Also studied is the effect of the strength of the interaction between the confined media's walls and the proteins. If the walls are only weakly or moderately attractive, a complex behavior emerges that depends on the size of the confining medium.

Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System

Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted tothe early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.

Bicelle size modulates the rate of bacteriorhodopsin folding.

The conformational equilibria of integral membrane proteins have proven extremely difficult to characterize within native lipid bilayers. To circumvent technical issues, investigations of the structure and stability of α-helical membrane proteins are often carried out in mixed micelle or bicelle solvents that mimic the membrane and facilitate measurements of reversible folding. Under these conditions, the energetics of membrane protein folding are typically proportional to the mole fraction of an anionic detergent in the micelle. However, investigations of the folding and unfolding of bacteriorhodopsin (bR) surprisingly revealed that the folding rate is also highly sensitive to the bulk molar concentration of lipids and detergents. We show here that this rate enhancement coincides with changes in bicelle size and suggest this effect arises through restriction of the conformational search space during folding. In conjunction with previous mutagenic studies, these results provide additional evidence that a topological search limits the rate of bR folding. Furthermore, this finding provides insights into the manner by which micellar and bicellar environments influence the conformational stability of polytopic membrane proteins.

Gene Therapy Strategies to Restore ER Proteostasis in Disease.

Proteostasis alterations are proposed as a transversal hallmark of several pathological conditions, including metabolic disorders, mechanical injury, cardiac malfunction, neurodegeneration, and cancer. Strategies to improve proteostasis aim to reduce the accumulation of specific disease-related misfolded proteins or bolster the endogenous mechanisms to fold and degrade abnormal proteins. Endoplasmic reticulum (ER) stress is a common pathological signature of a variety of diseases, which engages the unfolded protein response (UPR) as a cellular reaction to mitigate ER stress. Pharmacological modulation of the UPR is challenging considering the physiological importance of the pathway in various organs. However, local targeting of ER stress responses in the affected tissue using gene therapy is emerging as a possible solution to overcome side effects. The delivery of ER chaperones or active UPR components using adeno-associated virus (AAV) has demonstrated outstanding beneficial effects in several disease models (e.g., neurodegenerative conditions, eye disorders, and metabolic diseases). Here, we discuss current efforts to design and optimize gene therapy strategies to improve ER proteostasis in different disease contexts.

Hybrid biosensing cellulose‐based scaffolds for imaging‐assisted tissue engineering

3D tissue models closely mimic in vivo cell and tissue organization otherwise not seen in conventional cell culture. However, live cell imaging and quantification of cells growing within 3D scaffold are yet to be achieved for success of tissue engineering, cancer biology and regenerative medicine. Here, we aimed to design the environment‐sensitive scaffold material acting both as supporting material and a biosensor for cells growing in it. We chose cellulose‐binding domain of C. fimi CenA protein, termed as CBD (N‐terminal fragment, Ala32‐Thr137) and genetically fused it to either pH (enhanced cyan fluorescent protein, ECFP) or Ca2+ (gCaMP2)‐sensitive proteins. ECFP and gCaMP2 are prospective biosensors for quantitative Fluorescence Lifetime Imaging Microscopy (FLIM). Next, we optimised E. coli expression, purification and folding conditions for these chimeric proteins, achieving yields of ~6 mg/L culture, bright fluorescence (~55% folding rate) and sensitivity to extracellular pH and Ca2+.To assemble the functional biosensing scaffolds using CBD‐fusions, we performed labeling of the three different types of cellulose‐based materials: commercially available nanofibrillar cellulose Growdex™, bacterial cellulose spheres and decellularized plant tissues. Both CBD‐ECFP (pH‐sensitive) and CBD‐gCaMP2 (Ca2+‐sensitive) proteins efficiently labeled scaffolds in concentration‐dependent manner and remained stable under physiological conditions for > 7 days. In a proof‐of‐concept experiments, we tested usability of CBD‐ECFP‐stained Growdex scaffold as an extracellular pH sensor in FLIM: we observed robust and reversible changes in fluorescence lifetime (2.3–2.5 ns) over the range of physiological pH. Further, we tested the biosensing properties of CBD‐ECFP scaffolds, populated with cultured human colon cancer HCT116, mouse embryonic fibroblast (MEF) and primary stem cell‐derived mouse intestinal organoids. All the types of scaffolds were compatible with growth of cells with no evident toxicity, confirmed by experiments with unlabelled scaffolds.In conclusion, we have developed versatile labeling approach for producing biosensing cellulose‐based 3D scaffolds, suitable for different biosensors and types of cell and tissues, including single cells, spheroids and organoids. Such ‘hybrid’ biosensing scaffolds allow for real‐time measurement of extracellular biomolecules using quantitative FLIM microscopy, providing improvements in multi‐parametric imaging of engineered tissue models.Support or Funding InformationScience Foundation Ireland grant SFI 13/SIRG/2144.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Labeling for Quantitative Comparison of Imaging Measurements in Vitro and in Cells.

Qualitative imaging of biomolecular localization and distribution inside cells has revolutionized cell biology. Most of these powerful techniques require modifications to the target biomolecule. Over the past 10 years, these techniques have been extended to quantitative measurements, from in-cell protein folding rates to complex dissociation constants to RNA lifetimes. Such measurements can be affected even when a target molecule is just mildly perturbed by its labels. Here, the impact of labeling on protein (and RNA) structure, stability, and function in cells is discussed via practical examples from the recent literature. General guidelines for selecting and validating modification sites are provided to bring the best from cell biology and imaging to quantitative biophysical experiments inside cells.

Impact of hydrodynamic interactions on protein folding rates depends on temperature.

We investigated the impact of hydrodynamic interactions (HI) on protein folding using a coarse-grained model. The extent of the impact of hydrodynamic interactions, whether it accelerates, retards, or has no effect on protein folding, has been controversial. Together with a theoretical framework of the energy landscape theory (ELT) for protein folding that describes the dynamics of the collective motion with a single reaction coordinate across a folding barrier, we compared the kinetic effects of HI on the folding rates of two protein models that use a chain of single beads with distinctive topologies: a 64-residue α/β chymotrypsin inhibitor 2 (CI2) protein, and a 57-residue β-barrel α-spectrin Src-homology 3 domain (SH3) protein. When comparing the protein folding kinetics simulated with Brownian dynamics in the presence of HI to that in the absence of HI, we find that the effect of HI on protein folding appears to have a "crossover" behavior about the folding temperature. This means that at a temperature greater than the folding temperature, the enhanced friction from the hydrodynamic solvents between the beads in an unfolded configuration results in lowered folding rate; conversely, at a temperature lower than the folding temperature, HI accelerates folding by the backflow of solvent toward the folded configuration of a protein. Additionally, the extent of acceleration depends on the topology of a protein: for a protein like CI2, where its folding nucleus is rather diffuse in a transition state, HI channels the formation of contacts by favoring a major folding pathway in a complex free energy landscape, thus accelerating folding. For a protein like SH3, where its folding nucleus is already specific and less diffuse, HI matters less at a temperature lower than the folding temperature. Our findings provide further theoretical insight to protein folding kinetic experiments and simulations.

Life times of metastable states guide regulatory signaling in transcriptional riboswitches

Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. RNA folding pathways leading to functional ON and OFF regulation involve the formation of metastable states within well-defined sequence intervals during transcription. The kinetic requirements for the formation and preservation of these metastable states in the context of transcription remain unresolved. Here, we reversibly trap the previously defined regulatory relevant metastable intermediate of the Mesoplasma florum 2′-deoxyguanosine (2′dG)-sensing riboswitch using a photocaging-ligation approach, and monitor folding to its native state by real-time NMR in both presence and absence of ligand. We further determine transcription rates for two different bacterial RNA polymerases. Our results reveal that the riboswitch functions only at transcription rates typical for bacterial polymerases (10–50 nt s−1) and that gene expression is modulated by 40–50% only, while subtle differences in folding rates guide population ratios within the structural ensemble to a specific regulatory outcome.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "β-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer, including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the β-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel-phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA, and another model protein (OmpG) folded at significantly different rates and that an α-helix embedded inside the EspP β-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding.

Equilibrium and Non-equilibrium Studies of Protein G using High-Resolution Optical Trapping Unveil Heterogeneous Unfolding Pathways

Recent advances in spatial and temporal resolution of biophysical measurements have revealed ‘beyond two-state’ complexity in protein folding, even for small, single-domain proteins. In this work, the folding/unfolding kinetics of the B1 domain of inmmunoglobulin-binding protein G, a well-studied model system, was investigated using high-resolution optical traps. For non-equilibrium force ramp measurements, simple force-dependent folding distributions were observed. However, multiple peaks were seen in the unfolding force distribution, consistent with multiple parallel unfolding pathways or multiple folded states. We then performed long duration fixed trap measurements while slowly stepping the applied force. We were thereby able to directly measure folding and unfolding rate constants versus force under near equilibrium conditions. All molecules showed the same folding behavior, consistent with a simple two-state Bell's formula. However, these same molecules showed one of two unfolding behaviors at low forces, either ‘fast’ and force independent, or ‘slow’ and strongly force dependent. Equilibrium fixed trap observations of individual molecules for over tens of minutes show no switching between unfolding mechanisms amid hundreds of transitions. However, both unfolding mechanisms are observed for the same individual molecules using the force ramp method, indicating that out of equilibrium pulling may be necessary to switch individual molecule unfolding behavior. Taken together, these measurements indicate that protein G unfolds on a more complex energy landscape containing multiple pathways and intermediate states.

Increasing the stability of the articulated lorry at braking by locking the fifth wheel coupling

The jackknifing of the articulated lorry is determined by the loss of stability with respect to the vertical axis of the fifth wheel coupling, which can be caused by the failure of the brake system, the displacement of the center of mass of the semitrailer or tractor from the longitudinal axis of the vehicle, the road parameters (longitudinal and transverse slopes), the difference in the friction coefficients under the sides of the articulated lorry. In this regard, the issue of creating devices that prevent the jackknifing, and their control systems is important. A method is proposed for maintaining the stability of the movement of articulated lorry when braking both on a straight line and in a turn by blocking the relative rotation of the tractor and the trailer. Blocking occurs due to the creation of a stabilizing moment in the direction opposite to the angular rate of folding. To test the developed algorithm for locking the fifth wheel coupling, a mathematical model of the spatial motion of the articulated lorry was developed, including the models of interaction of an elastic tire with a rigid terrain, suspension systems, transmission, steering, fifth-wheel coupling. The efficiency and effectiveness of the coupling locking control system is proved by comparing the results of the simulation of a straight-line braking and braking in turn. It is shown that the application of the control system significantly increases the stability of the road train.