Folding Intermediate State Research Articles

DeFNet: deconstructed strategy for multi-step fabric folding tasks

Fabric folding through robots is complex and challenging due to the deformability of the fabric. Based on the deconstruction strategy, we split the tough multi-step fabric folding task into three relatively simple sub-tasks and propose a Deconstructed Fabric Folding Network (DeFNet), including three corresponding modules to solve them. (1) We use the Folding Planning Module (FPM), which is based on the Latent Space Roadmap, to infer the shortest folding intermediate states from the start to the goal in latent space. (2) We utilize the flow-based approach, Folding Action Module (FAM), to calculate the action coordinates and execute them to reach the inferred intermediate state. (3) We introduce an Iterative Interactive Module (IIM) for multi-step fabric folding tasks, which can iteratively execute the FPM and FAM after every grasp-and-place action until the fabric reaches the goal. Experimentally, we demonstrate our method on fabric folding tasks against three baselines in simulation. We also apply the method to an existing robotic system and present its performance.

Stabilization of Non-Native Folds and Programmable Protein Gelation in Compositionally Designed Deep Eutectic Solvents.

Proteins are adjustable units from which biomaterials with designed properties can be developed. However, non-native folded states with controlled topologies are hardly accessible in aqueous environments, limiting their prospects as building blocks. Here, we demonstrate the ability of a series of anhydrous deep eutectic solvents (DESs) to precisely control the conformational landscape of proteins. We reveal that systematic variations in the chemical composition of binary and ternary DESs dictate the stabilization of a wide range of conformations, that is, compact globular folds, intermediate folding states, or unfolded chains, as well as controlling their collective behavior. Besides, different conformational states can be visited by simply adjusting the composition of ternary DESs, allowing for the refolding of unfolded states and vice versa. Notably, we show that these intermediates can trigger the formation of supramolecular gels, also known as eutectogels, where their mechanical properties correlate to the folding state of the protein. Given the inherent vulnerability of proteins outside the native fold in aqueous environments, our findings highlight DESs as tailorable solvents capable of stabilizing various non-native conformations on demand through solvent design.

The Role of Molecular Chaperones in Polypeptide Folding

Purpose: The aim of the study is to examine the role of molecular chaperones in polypeptide.
 Methodology: This study adopted a desktop methodology. This study used secondary data from which include review of existing literature from already published studies and reports that was easily accessed through online journals and libraries.
 Findings: The study revealed that molecular chaperones assist in the folding of polypeptides by stabilizing intermediate folding states and preventing the aggregation of unfolded or misfolded proteins. Molecular chaperones are crucial in preventing protein misfolding and aggregation-associated diseases, particularly in the context of neurodegenerative disorders
 Unique Contribution to Theory, Practice and Policy: The study was anchored on The Kinetic Partitioning Model was Proposed by Susan L. Lindquist and Levinthal's Paradox and the Hierarchical Folding Model and was proposed by Cyrus Levinthal. The study recommended support funding initiatives and research grants that focus on investigating the role of molecular chaperones in protein folding and their implications in various fields, including biotechnology, medicine, and drug discovery. Promote interdisciplinary research efforts to address the challenges and opportunities associated with chaperone-mediated folding.

Structural basis of plp2-mediated cytoskeletal protein folding by TRiC/CCT.

The cytoskeletal proteins tubulin and actin are the obligate substrates of TCP-1 ring complex/Chaperonin containing TCP-1 (TRiC/CCT), and their folding involves co-chaperone. Through cryo-electron microscopy analysis, we present a more complete picture of TRiC-assisted tubulin/actin folding along TRiC adenosine triphosphatase cycle, under the coordination of co-chaperone plp2. In the open S1/S2 states, plp2 and tubulin/actin engaged within opposite TRiC chambers. Notably, we captured an unprecedented TRiC-plp2-tubulin complex in the closed S3 state, engaged with a folded full-length β-tubulin and loaded with a guanosine triphosphate, and a plp2 occupying opposite rings. Another closed S4 state revealed an actin in the intermediate folding state and a plp2. Accompanying TRiC ring closure, plp2 translocation could coordinate substrate translocation on the CCT6 hemisphere, facilitating substrate stabilization and folding. Our findings reveal the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the biogenesis machinery TRiC and plp2 and extend our understanding of the links between cytoskeletal proteostasis and related human diseases.

Measuring DNA extension during unfolding in a centrifugal force microscope

DNA in sperm cells can form compact toroids when bound with proteins from the protamine family. To study DNA folding and toroid formation, we have built a centrifugal force microscope (CFM) to unfold DNA and observe the intermediate folding states. In a CFM, micron-sized beads tethered to the sample by a DNA strand are placed into a microscope within a desktop centrifuge. The centrifuge applies a centrifugal force (a pseudoforce caused by inertia) to our sample, unfolding the DNA. Recording and analyzing the bead motion can yield information on the extension of the DNA over time and the intermediate stages of DNA condensation. Our CFM is operational at low centrifuge speeds, but we did not have a method for measuring the extension of the DNA. Here, we will discuss our progress in creating a program that gathers and analyzes video microscopy data from the CFM to measure extension in real time. This program will be useful in collecting data on the pathway of DNA condensation and for other force spectroscopy measurements with the CFM.

Transient On- and Off-Pathway Protein Folding Intermediate States Characterized with NMR Relaxation Dispersion.

The earliest events in the folding of a protein are in general poorly understood. We used NMR R2 relaxation dispersion experiments to study transient local collapse events in the unfolded-state (U) conformational ensemble of apomyoglobin (apoMb). Local residual secondary structure (seen in regions corresponding to the A, D, E, and H helices of the folded protein) is largely unchanged over the pH range of 2.3-2.75, yet a significant pH-dependent increase in the conformational exchange contribution to the R2 relaxation rate (Rex) indicates that transient intramolecular contacts occur on a microsecond to millisecond time scale at pH 2.75. A comparison of 15N and 13CO relaxation dispersion data at pH 2.75 for residues in the A, B, G, and H regions, which participate in the earliest folding intermediates, indicates that chain collapse and secondary structure formation are rapid and concomitant. Increasingly stabilizing conditions (lower temperature, higher pH) result in the observation of a relaxation dispersion in the C, CD, and E regions of the protein, which are known to fold at later stages. Mutation of Trp14 in the A-helix region to Ala eliminates conformational exchange throughout the protein, and the mutation of hydrophobic residues in other regions results in the selective inhibition of conformational exchange in the B, G, or H regions. The R2 dispersion data for WT apoMb at pH 2.75 and 10 °C are best fit to a four-state model ABGH ⇆ AGH ⇆ U ⇆ ABCD that includes on-pathway (AGH and ABGH) and off-pathway (ABCD) transiently folded states, both of which are required to explain the behavior of the mutant proteins. The off-pathway intermediate is destabilized at higher temperatures. Our analysis provides insights into the earliest stages of apoMb folding where the collapsing polypeptide chain samples both productive and nonproductive states with stabilized secondary structure.

A single molecule investigation of i-motif stability, folding intermediates, and potential as in-situ pH sensor.

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for low pH levels (pH < 6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Förster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the intermediate states visited during i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor to establish the time scale for the pH drop in a commonly used oxygen scavenging system.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

Folding intermediate states of the parallel human telomeric G-quadruplex DNA explored using Well-Tempered Metadynamics

An increasingly comprehension of the folding intermediate states of DNA G-quadruplexes (G4s) is currently an important scientific challenge, especially for the human telomeric (h-tel) G4s-forming sequences, characterized by a highly polymorphic nature. Despite the G-triplex conformation was proposed as one of the possible folding intermediates for the antiparallel and hybrid h-tel G4s, for the parallel h-tel topology with an all-anti guanine orientation, a vertical strand-slippage involving the G-triplets was proposed in previous works through microseconds-long standard molecular dynamics simulations (MDs). Here, in order to get further insights into the vertical strand-slippage and the folding intermediate states of the parallel h-tel G4s, we have carried out a Well-Tempered Metadynamics simulation (WT-MetaD), which allowed us to retrieve an ensemble of six G4s having two/G-tetrad conformations derived by the G-triplets vertical slippage. The insights highlighted in this work are aimed at rationalizing the mechanistic characterisation of the parallel h-tel G4 folding process.

Zig Zag AFM Protocol Reveals New Intermediate Folding States of Bacteriorhodopsin

Zig Zag AFM Protocol Reveals New Intermediate Folding States of Bacteriorhodopsin

A transient absence of SMC-mediated loops after mitosis.

After mitosis chromosomes are drastically reshaped. A study now charts the dynamics of this conformational change at high temporal resolution. During the shift from one loop-forming complex (condensin) to another (cohesin), an intermediate chromosome folding state exists in which neither of these complexes are associated with chromatin.

Folding Mechanism of β-Helical Passenger Domains from a Bacterial Autotransporter

Virulence factors, small peptides attached to larger autotransporter proteins, are a common source of virulence in infectious, Gram-negative bacteria and a new focus for antimicrobial drug development. While the transportation of the virulence factor across the inner membrane is well-studied, the mechanism of how these peptides are secreted through the outer membrane remains unclear due to the absence of traditional energy sources, such as ATP or ion gradients, at the outer membrane. Interestingly, many of these proteins come attached to large β-helical passenger domains, which do not appear to be participating in any of their virulence behavior. In addition, folding of the β-helix occurs significantly faster in vivo than in vitro, so the passenger domain likely folds along a faster, vectorial pathway as its being secreted through the outer membrane. Experimental results show that the relative stability at the N- and C-terminus of the passenger domain largely affects the secretion rate of these proteins, suggesting that the folding of the β-helix structure plays an important role in efficient secretion. In this study, we used three different simulation methods to investigate the folding mechanism and kinetics of the passenger domain of Pertactin, an autotransporter from Bordetella pertussis. Multidimensional replica-exchange umbrella sampling simulations reveal how cooperative folding beginning at the C-terminus enhances the kinetics of folding, while steered molecular dynamics simulations show differences in mechanical responses at the N- and C-terminus. Lastly, we use Markov state modelling to find intermediate folding states as well as possible misfolded structures, which may act as kinetic traps.

Monitoring Unfolding of Titin I27 Single and Bi Domain with High-Pressure NMR Spectroscopy

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular dynamics (MD) has often been combined with force spectroscopy experiments to decipher the unfolding mechanism of titin immunoglobulin-like single or multidomain, the giant multimodular protein from sarcomeres, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single domain and tandem. Because this method brings residue-specific information on the folding process, it can provide quasiatomic details on this process without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the combination of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB β-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the A′G parallel β-sheet of the β-sandwich has been confirmed as the Achilles heel of the three-dimensional scaffold: its disruption yields complete unfolding with very similar characteristics (free energy, unfolding volume, kinetics rate constants) for the two constructs.

Proline and Water Stabilization of a Universal Two-Step Folding Mechanism for β-Turn Formation in Solution.

The atomic scale process by which proteins fold into their functional forms in aqueous solutions is still not well understood. While there is clearly an interplay between the sequence of the protein and the surrounding water solvent that leads to highly specific and reproducible folding in nature, there is still an ongoing debate concerning how water molecules aid in driving the folding process. By using a combination of techniques that provide information at the atomic level-neutron and X-ray diffraction and computer simulations-the mechanism of folding in a series of peptides that only vary with respect to the central side-chain residue has been determined. Specifically, β-turn formation for the KGXGK peptide (where X = P, G, S or L) occurs via a two-step water-driven attraction between specific sites on the peptide backbone. This proposed mechanism suggests that the site-specific hydration of the backbone facilitates the initial stages of protein folding and that this hydration interaction in combination with the presence of proline in the i + 1 position helps to stabilize the folded and intermediate folding state of the peptide in solution, leading to a greater propensity for PG containing sequences to occur in β-turns in proteins.

Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp

Protein folding process involves formation of transiently occurring intermediates that are difficult to isolate and characterize. It is both necessary and interesting to characterize the structural conformations adopted by these intermediates, also called molten globules (MG), to understand protein folding. Here, we investigated the equilibrium (un)folding intermediate state of T4 phage gene product 45 (gp45, also known as DNA polymerase processivity factor or sliding clamp) obtained during chemical denaturation. We show that gp45 undergoes substantial conformational rearrangement during unfolding and forms an expanded dry-MG. By monitoring the fluorescence of tryptophans that were strategically introduced at various sites, we demonstrate that the urea-treated molecule has its surface residues flip inside the core, and closely placed residues move farther. We were also able to isolate and purify the MG form of gp45 in native condition (i.e., nondenaturing buffer, at physiological pH and temperature); characteristics of this purified molecule substantially match with urea-treated wild-type gp45. To the best of our knowledge, this is one of the few reports that demonstrate the isolation and purification of a protein folding intermediate in native condition. We believe that our work not only allows us to dissect the process of protein folding, but will also help in the designing of folding inhibitors against sliding clamps to treat a wide variety of diseases from bacterial infection to cancer, due to the vast presence of clamps in all the domains of life.

Directing folding pathways for multi-component DNA origami nanostructures with complex topology

Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

Existence of molten globule state in homocysteine-induced protein covalent modifications.

Homocysteine thiolactone is a toxic metabolite produced from homocysteine by amino-acyl t-RNA synthetase in error editing reaction. The basic cause of toxicity of homocysteine thiolactone is believed to be due to the adduct formation with lysine residues (known as protein N-homocysteinylation) leading to protein aggregation and loss of enzyme function. There was no data available until now that showed the effect of homocysteine thiolactone on the native state structural changes that led to aggregate formation. In the present study we have investigated the time dependent structural changes due to homocysteine thiolactone induced modifications on three different proteins having different physico-chemical properties (cytochrome-c, lysozyme and alpha lactalbumin). We discovered that N-homocysteinylation leads to the formation of molten globule state—an important protein folding intermediate in the protein folding pathway. We also found that the formation of the molten globule state might be responsible for the appearance of aggregate formation. The study indicates the importance of protein folding intermediate state in eliciting the homocysteine thiolactone toxicity.

Measuring hydrogen exchange in proteins by selective water saturation in (1)H- (15)N SOFAST/BEST-type experiments: advantages and limitations.

HET(ex)-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199-211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provide a better resolution at the expense of some loss in sensitivity. We discuss the theoretical background and implementation of the experiment, and demonstrate its performance for an intrinsically disordered protein, 2 well folded globular proteins, and a transiently populated folding intermediate state. We also provide a critical evaluation of the level of accuracy that can be obtained when extracting quantitative exchange rates from HET(ex) NMR measurements.

An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.

Decrease in RNA Folding Cooperativity by Deliberate Population of Intermediates in RNA G‐Quadruplexes

Keeping a broad (RNA) perspective: conventional biochemical detection systems only have a 100-fold linear response range. The range of potassium concentrations detected by an RNA G-quadruplex sequence can be broadened by intentionally populating multiple intermediate folding states. The folding of the RNA G-quadruplexes was monitored by both circular dichroism and intrinsic fluorescence spectroscopy.