Complex Folding Research Articles

Insights into the subsurface structure of wrinkle ridges on Mars

Wrinkle ridges are important landforms on Mars and other planetary bodies and result from horizontal tectonic shortening. This study provides insights into the subsurface of selected wrinkle ridge sites, based on exposures provided by steep escarpments and crater slopes that crosscut wrinkle ridges. We mapped the complex fold and fault patterns and measured fault dips, where faults appear planar. The fault pattern underneath wrinkle ridges is diverse and is dominated by reverse faults and thrusts. The predominance of one fault system leads to morphological asymmetries of many wrinkle ridges. The upper tip of the dominant thrust fault is commonly situated beneath the steeper wrinkle ridge slope and may outcrop at its base. Polarity changes of wrinkle ridges along strike go along with a change of the dominant thrust direction. Symmetric wrinkle ridges are formed when a conjugate system of thrusts exists in the subsurface. Several wrinkle ridges display a main thrust fault whose dip angle bends over to a shallower dip at depth. The measurements of average dip for primary thrust and backthrust faults are 37° ± 2° and 28 ± 2°, respectively. In general, most of the examined wrinkle ridges are characterized by a multitude of subsidiary and backthrust faults. With the limitation that the uppermost 500-1000 m of the wrinkle ridges subsurface are accessible in this study and despite the large morphological and structural variability of wrinkle ridges, the kinematics of fault-propagation folds models seem to show a large match to our observations and measurements. Likewise, continuum mechanics models show a good fit to the obtained structural data. Fault-bend fold models and simple buckle folding models seem less compatible with observations.

Deciphering the Folding Mechanism of Proteins G and L and Their Mutants.

Much of our understanding of folding mechanisms comes from interpretations of experimental ϕ and ψ value analysis, relating the differences in stability of the transition state ensemble (TSE) and folded state. We introduce a unified approach combining simulations and Bayesian inference to provide atomistic detail for the folding mechanism of proteins G and L and their mutants. Proteins G and L fold to similar topologies despite low sequence similarity, but differ in their folding pathways. A fast folding redesign of protein G, NuG2, switches folding pathways and folds through a similar pathway with protein L. A redesign of protein L also leads to faster folding, respecting the original folding pathway. Our Bayesian inference approach starts from the same prior on all systems and correctly identifies the folding mechanism for each of the four proteins, a success of the force field and sampling strategy. The approach is computationally efficient and correctly identifies the TSE and intermediate structures along the folding pathway in good agreement with experiments. We complement our findings by using two orthogonal approaches that differ in computational cost and interpretability. Adaptive sampling MD combined with the Markov state model provides a kinetic model that confirms the more complex folding mechanism of protein G and its mutant. Finally, a novel fragment decomposition approach using AlphaFold identifies preferences for secondary structure element combinations that follow the order of events observed in the folding pathways.

A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein

Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using a member of a structurally uncharacterized, eukaryotic receptor family as an example we employ hydrogen–deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify a minimal expression construct that can be produced with high yields and moderate costs.

Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.

Multi-scale visualization of incipient CaCO3 scaling on the polyamide layer of reverse osmosis membranes

Although robust models on water transport across reverse osmosis (RO) membranes exist, detailed mechanistic insights of mineral scaling in the active polyamide (PA) layer, especially through visual investigations, are still lacking. Herein, a holistic approach of imaging the onset of mineral scales in the PA-layer is detailed at different length scales. BW30 RO membranes were scaled by running CaCO3-rich feedwater in a narrow, spacer-filled crossflow channel for 120 min. Then, microscopy and spectroscopy-based characterization tools were applied to investigate the CaCO3 scaling in the PA-layer. The vaterite phase of CaCO3 dominated at the incipient stage and was amplified at higher flux. TEM imaging, followed by digital staining with unsupervised K-means clustering, demonstrated incipient scaling primarily on top of the intricate folds of the PA-layer. Scales with a characteristic size between 1 and 6 nm were also identified deep inside the randomly distributed microvoids formed by those folds, hinting at heterogeneous crystallization of CaCO3. Thus, membranes designed to limit the complex folding of the PA-layer could show more scale resistance. The improved visualization of PA-layer via TEM–K-means and resulting insights can be broadly used in designing novel scale-resistant membranes and evaluating the efficacy of membrane cleaning operations.

AlphaFold predicts the most complex protein knot and composite protein knots.

The computer artificial intelligence system AlphaFold has recently predicted previously unknown three‐dimensional structures of thousands of proteins. Focusing on the subset with high‐confidence scores, we algorithmically analyze these predictions for cases where the protein backbone exhibits rare topological complexity, that is, knotting. Amongst others, we discovered a 71‐knot, the most topologically complex knot ever found in a protein, as well several six‐crossing composite knots comprised of two methyltransferase or carbonic anhydrase domains, each containing a simple trefoil knot. These deeply embedded composite knots occur evidently by gene duplication and interconnection of knotted dimers. Finally, we report two new five‐crossing knots including the first 51‐knot. Our list of analyzed structures forms the basis for future experimental studies to confirm these novel‐knotted topologies and to explore their complex folding mechanisms.

Pulse labeling reveals the tail end of protein folding by proteome profiling.

Accurate and efficient folding of nascent protein sequences into their native states requires support from the protein homeostasis network. Herein we probe which newly translated proteins are thermo-sensitive, making them susceptible to misfolding and aggregation under heat stress using pulse-SILAC mass spectrometry. We find a distinct group of proteins that is highly sensitive to this perturbation when newly synthesized but not once matured. These proteins are abundant and highly structured. Notably, they display a tendency to form β sheet secondary structures, have more complex folding topology, and are enriched for chaperone-binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides are also more often components of stable protein complexes in comparison with other proteins. Combining these findings suggests the existence of a specific subset of proteins in the cell that is particularly vulnerable to misfolding and aggregation following synthesis before reaching the native state.

Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTβ) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

Characterization of early and late transition states of the folding pathway of a SH2 domain.

Albeit SH2 domains are abundant protein–protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C‐terminal SH2 domain of SHP2, conducted through a combination of site‐directed mutagenesis and kinetic (un)folding experiments (Φ‐value analysis). The energetic profile of the folding reaction of the C‐SH2 domain is described by a three‐state mechanism characterized by the presence of two transition states and a high‐energy intermediate. The production of 29 site‐directed variants allowed us to calculate the degree of native‐like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ‐values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N‐terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.

The TRiCky Business of Protein Folding in Health and Disease.

Maintenance of the cellular proteome or proteostasis is an essential process that when deregulated leads to diseases like neurological disorders and cancer. Central to proteostasis are the molecular chaperones that fold proteins into functional 3-dimensional (3D) shapes and prevent protein aggregation. Chaperonins, a family of chaperones found in all lineages of organisms, are efficient machines that fold proteins within central cavities. The eukaryotic Chaperonin Containing TCP1 (CCT), also known as Tailless complex polypeptide 1 (TCP-1) Ring Complex (TRiC), is a multi-subunit molecular complex that folds the obligate substrates, actin, and tubulin. But more than folding cytoskeletal proteins, CCT differs from most chaperones in its ability to fold proteins larger than its central folding chamber and in a sequential manner that enables it to tackle proteins with complex topologies or very large proteins and complexes. Unique features of CCT include an asymmetry of charges and ATP affinities across the eight subunits that form the hetero-oligomeric complex. Variable substrate binding capacities endow CCT with a plasticity that developed as the chaperonin evolved with eukaryotes and acquired functional capacity in the densely packed intracellular environment. Given the decades of discovery on the structure and function of CCT, much remains unknown such as the scope of its interactome. New findings on the role of CCT in disease, and potential for diagnostic and therapeutic uses, heighten the need to better understand the function of this essential molecular chaperone. Clues as to how CCT causes cancer or neurological disorders lie in the early studies of the chaperonin that form a foundational knowledgebase. In this review, we span the decades of CCT discoveries to provide critical context to the continued research on the diverse capacities in health and disease of this essential protein-folding complex.

Engineering protein activity into off-the-shelf DNA devices

SummaryDNA-based devices are straightforward to design by virtue of their predictable folding, but they lack complex biological activity such as catalysis. Conversely, protein-based devices offer a myriad of functions but are much more difficult to design due to their complex folding. This study combines DNA and protein engineering to generate an enzyme that is activated by a DNA sequence of choice. A single protein switch, engineered from nanoluciferase using the alternate-frame-folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for specific nucleic acid sequences, sensors for serotonin and ATP, and a two-input logic gate. nLuc-AFF is a genetically encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by a phone camera. nLuc-AFF retains ratiometric readout in 100% serum, making it suitable for analyzing crude samples in low-resource settings. This approach can be applied to other proteins and enzymes to convert them into DNA-activated switches.

Ligands with polyfluorophenyl moieties promote a local structural rearrangement in the Spinach2 and Broccoli aptamers that increases ligand affinities.

The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand how nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI [(Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one] that reach out from the Spinach2 ligand binding pocket. Studies of the interaction of these compounds with the aptamers revealed that polyfluorophenyl-modified ligands initiate a slow change in aptamer affinity that takes an extended time (half-life of ∼40 min) to achieve. The change in affinity appears to involve an initial disruption of the entrance to the ligand binding pocket followed by a gradual transition to a more defined structure for which the most likely driving force is an interaction of the gateway adenine with a nearby 2'OH group. These results suggest that polyfluorophenyl modifications might increase the ability of small molecule drugs to disrupt local structure and promote RNA remodeling.

CENP-B-mediated DNA loops regulate activity and stability of human centromeres

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction andclustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope.

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

Site-specific integration as an efficient method for production of recombinant human hyaluronidase PH20 in semi-adherent cells.

PH20 is a hyaluronidase enzyme that can hydrolyze the glycosidic bond in hyaluronic acid as the major proteoglycan found in extracellular matrices. In the present study, we constructed and characterized two donor plasmids, one of them with one and the second with two PH20 expression cassettes. The expression vectors were site specifically integrated into the genome of HEK293T cells using PhiC31 integrase system to develop HEK293T stable cell lines secreting His-tagged recombinant human PH20 (rhPH20) in the culture supernatant. The produced rhPH20 was quantified using ELISA and turbidimetric assay tests, and its catalytic activity was also assessed by treating the mouse cumulus-oocyte complexes. Our results showed that the secreted rhPH20 in the culture supernatant had the specific activity of 16,660IU/mg and the recombinant enzyme was able to remove the cumulus cells from oocytes. The results also indicated that phiC31 enzyme inserted the PH20-expressing donor vectors into the specific pseudo attP sites including 10q21.2 and 20q11.22 in the genome of the target cells with different copy numbers. Taken together, our findings demonstrate that PhiC31 integrase system is able to be applied as a robust tool for efficient production and secretion of soluble and active rhPH20 by HEK293T cells as a semi-adherent human cell line. KEY POINTS: • Efficient production of human recombinant PH20 in a semi-adherent human cell line • Successful application of PhiC31 integrase system for generation of stable recombinant clones • Use of a human cell line for expression of a recombinant human protein due to complex and efficient post-translational modifications and protein folding.

What Have We Learned from Design of Function in Large Proteins?

The overarching goal of computational protein design is to gain complete control over protein structure and function. The majority of sophisticated binders and enzymes, however, are large and exhibit diverse and complex folds that defy atomistic design calculations. Encouragingly, recent strategies that combine evolutionary constraints from natural homologs with atomistic calculations have significantly improved design accuracy. In these approaches, evolutionary constraints mitigate the risk from misfolding and aggregation, focusing atomistic design calculations on a small but highly enriched sequence subspace. Such methods have dramatically optimized diverse proteins, including vaccine immunogens, enzymes for sustainable chemistry, and proteins with therapeutic potential. The new generation of deep learning-based ab initio structure predictors can be combined with these methods to extend the scope of protein design, in principle, to any natural protein of known sequence. We envision that protein engineering will come to rely on completely computational methods to efficiently discover and optimize biomolecular activities.

Electrochemically driven assembly of framework nucleic acids

• The utilization of an electrochemical chip to synthesis DNA nanostructures was firstly developed. • Tetrahedron DNA nanostructures of different sizes (20 bp, 26 bp and 37 bp) and octahedral DNA nanostructure were successfully assembled within 6 min. • The prepared DNA nanostructures exhibit the same structural characteristics and electrochemical performance in comparison to the ones fabricated with thermal annealing method. • The relations between electric potential and pH value were built. Electrochemically controlled synthesis of small molecules, DNA and various nanostructures provided unparalleled advantages to remotely control chemical reactions with high spatial and temporal precision. Although electrochemistry has been reported to actuate the reversible movement of DNA switch or tune the formation of hydrogen bonds in double strand DNA, electrochemically driven formation of DNA nanostructures remains a challenge as the result of their three-dimensional shapes and complex DNA folding pathway. Herein, through integrating a microfabricated chip with three-electrode system, we reported an electrochemical method to control the formation of tetrahedral DNA nanostructures. Using this method, the relation between the potential and solution pH value, and the melting temperature of double strand DNA was built, which facilitates synthesis of the tetrahedron DNA nanostructures in 6 min. Structure characterization of the prepared DNA nanostructures with atomic force microscopy (AFM) proved the physical integrity of the prepared DNA structures. By modifying the prepared DNA structure on the gold electrodes, a DNA nanostructure-based electrochemical biosensor with high sensitivity toward target DNA was fabricated. Based on these advantages, this method holds promise to develop high-throughput electrochemical microdevices capable of in-situ production of DNA nanostructures and the consequent multichannel biosensing.

Resolving the fine structure in the energy landscapes of repeat proteins.

Ankyrin (ANK) repeat proteins are coded by tandem occurrences of patterns with around 33 amino acids. They often mediate protein-protein interactions in a diversity of biological systems. These proteins have an elongated non-globular shape and often display complex folding mechanisms. This work investigates the energy landscape of representative proteins of this class made up of 3, 4 and 6 ANK repeats using the energy-landscape visualisation method (ELViM). By combining biased and unbiased coarse-grained molecular dynamics AWSEM simulations that sample conformations along the folding trajectories with the ELViM structure-based phase space, one finds a three-dimensional representation of the globally funnelled energy surface. In this representation, it is possible to delineate distinct folding pathways. We show that ELViMs can project, in a natural way, the intricacies of the highly dimensional energy landscapes encoded by the highly symmetric ankyrin repeat proteins into useful low-dimensional representations. These projections can discriminate between multiplicities of specific parallel folding mechanisms that otherwise can be hidden in oversimplified depictions.

From Dirca to design: printmaking with leatherwood (Dirca mexicana) bark paper

ABSTRACT Washi is paper made by hand from the bark of native Japanese shrubs. Washi is a common medium used for printmaking and paper crafts. Artists who have studied nagashi-zuki, a sheet-forming method unique to washi, often import Japanese fibers because alternatives with similar properties have not been identified. We propose Dirca L. (leatherwood), a shrub endemic to North America, as a source of fibers with properties similar to those plants traditionally used to make washi. The thinness and strength of the leatherwood paper allows it to withstand repeated bending, folding and creasing better than paper made from species of Wikstroemia (Japanese fiber), suggesting an alternative for use with various printmaking techniques and paper arts and crafts that involve folding, such as origami. We engaged printmakers and origami artists in creating original pieces using our leatherwood paper and evaluated how the paper responds to various printmaking techniques and complex folding. We identified Dirca mexicana as a source of fibers with similar properties to species of Wikstroemia used to make gampi washi. Handmade D. mexicana bark paper was successfully used as a paper medium for intaglio, lithography, relief, digital, and screenprinting printmaking techniques, as well as, complex folding origami sculptures.

Untethered Origami Worm Robot with Diverse Multi-Leg Attachments and Responsive Motions under Magnetic Actuation

Nowadays, origami folding in combination with actuation mechanisms can offer deployable structure design, yield compliance, and have several properties of soft material. An easy complex folding pattern can yield an array of functionalities in actuated hinges or active spring elements. This paper presents various cylinder origami robot designs that can be untethered magnetically actuated. The different designs are analyzed and compared to achieve the following three types of motion: Peristaltic, rolling, and turning in different environments, namely, board, sandpaper, and sand. The proposed origami robot is able translate 53 mm in peristaltic motion within 20 s and is able to roll one complete cycle in 1 s and can turn ≈180∘ in 1.5 s. The robot also demonstrated a peristaltic locomotion at a speed of ≈2.5 mm s−1, ≈1.9 mm s−1, and ≈1.3 mm s−1 in board, sandpaper, and sand respectively; rolling motion at a speed of 1 cycle s−1, ≈0.66 cycles s−1, and ≈0.33 cycles s−1 in board, sandpaper, and sand respectively; and turning motion of ≈180∘, ≈83∘, and ≈58∘ in board, sandpaper, and sand respectively. The evaluation of the robotic motion and actuation is discussed in detail in this paper.