Folding Units Research Articles

Exploring structural diversity across the protein universe with The Encyclopedia of Domains.

The AlphaFold Protein Structure Database (AFDB) contains more than 214 million predicted protein structures composed of domains, which are independently folding units found in multiple structural and functional contexts. Identifying domains can enable many functional and evolutionary analyses but has remained challenging because of the sheer scale of the data. Using deep learning methods, we have detected and classified every domain in the AFDB, producing The Encyclopedia of Domains. We detected nearly 365 million domains, over 100 million more than can be found by sequence methods, covering more than 1 million taxa. Reassuringly, 77% of the nonredundant domains are similar to known superfamilies, greatly expanding representation of their domain space. We uncovered more than 10,000 new structural interactions between superfamilies and thousands of new folds across the fold space continuum.

Improved constructs for bait RNA display in a bacterial three-hybrid assay.

We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.

Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in β-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

Assembly law of multi-ring hybrid origami mechanism and calculation method of the degree of freedom

This paper proposes a method for calculating the degrees of freedom of multi-loop hybrid folding mechanisms, which is used to calculate the degrees of freedom of multi-loop hybrid folding mechanisms, redefines the problem of not being able to determine whether the folding units are in series and parallel during the assembly process, summaries the two cases of redundant constraints, gives the calculation principle of multi-loop hybrid folding mechanisms, calculates the degrees of freedom for The calculation of degrees of freedom for the assembly of a variety of different crease folding units is carried out to verify the accuracy and feasibility of the degree of freedom calculation rule proposed in this paper. The control of the hand claw is improved and the number of drives added to the envelope type paper folding hand claw to capture space debris is reduced, greatly improving the reliability and stability of the paper-folding hand claw in space capture work, which is of great significance to the problem of calculating degrees of freedom after the assembly of paper folding units and the analysis of degrees of freedom of complex paper folding mechanisms and their design.

Photo-Responsive Dynamic Organic Room-Temperature Phosphorescence Materials Based on a Functional Unit Combination Strategy.

A rational molecular design strategy facilitates the development of a purely organic room-temperature phosphorescence (RTP) material system with precisely regulated luminescence properties, which surely promotes its functional integration and intelligent application. Here, a functional unit combination strategy is proposed to design novel RTP molecules combining a folding unit with diverse luminescent cores. The different luminescent cores are mainly responsible for tunable RTP properties, while the folding unit contributes to the spin-orbit coupling (SOC) enhancement, which makes the RTP material design as workable as the building block principle. By this strategy, a series of color/lifetime-tunable RTP materials is achieved with unique photo-responsive RTP enhancement when subjected to UV irradiation, which expands their application scenarios in reusable privacy tags, advanced "4D" encryption, and phase separation analysis of blended polymers. This work suggests a simple and effective strategy to design purely organic RTP materials with tunable color and lifetime, and also provides new application options for photo-responsive dynamic RTP materials.

The Ser7 of RNA Pol II-CTD influences the recruitment of Cdc73 for mRNA transcription

The carboxyl terminal domain of the largest subunit of eukaryotic RNA polymerase II (RNAPII) consists of highly conserved tandem repeats of Tyr1Ser2Pro3Thr4Ser5Pro6Ser7, referred as CTD. The CTD undergoes posttranslational modifications where the interplay of kinases imparts specific CTD phosphorylations, recognized by regulatory proteins that help in the mRNA transcription. Here, the Ser5 phosphorylation (Ser5P) remains high during the transcription initiation, followed by the Ser2P which peaks towards the termination and the Ser7P remains high throughout the transcription process. The Paf1 elongation complex (Paf1C) through its Cdc73 subunit is recruited to the phosphorylated CTD and play active role during different stages of mRNA transcription. We show that the CTD binding domain of Cdc73 is an independent folding unit which interacts with the hyper phosphorylated CTD. The 500 ns MD simulation studies further identified the binding interface and the pattern of CTD phosphorylation involved in the interaction with Cdc73. The possible key residues were mutated and the subsequent pull down analysis suggests that the phosphorylated Ser2, Ser5 and Ser7 of the tandem CTD heptads interact respectively with Arg310, Arg268 and Arg300 of Cdc73. Our finding provides new insight for Cdc73 function during mRNA transcription.

The Saccharomyces cerevisiae SR protein Npl3 interacts with hyperphosphorylated CTD of RNA Polymerase II

The catalytic subunit of RNA Polymerase II contains a highly conserved carboxy terminal domain (CTD) composed of multiple tandem heptad sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. The non-proline residues in CTD undergo posttranslational modifications, with Ser5 phosphorylation (Ser5P) predominating at the start of the transcription cycle and Ser2P at the end, while other phosphorylation levels are high all throughout. The differentially phosphorylated CTD is recognized by regulatory proteins, helpful during mRNA transcription and export. One such protein Npl3 is composed of two RNA binding domains and a C-terminus RGG/SR domain. The Ser411 of Npl3 is reported to make direct contact with Ser2P of CTD for its recruitment and function, while the Npl3 lacking of C-terminal 25 amino acids (Npl3Δ389–414) showed no apparent defects in mRNA synthesis. Here, we report that the RNA binding domains of Npl3 are separate folding units and interact also with the CTD. The interaction between Npl3 and CTD appears to involve not just Ser2P, but also the Ser5P and Ser7P. The Arg126 of the first RNA binding domain interacts with Ser2P whereas the Arg235 of the second RNA binding domain interacts with either Ser7P or Ser5P of another heptad. The finding provides new insight of Npl3 function for mRNA transcription.

Assembly landscape for the bacterial large ribosomal subunit

Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.

AlphaFold-predicted protein structures and small-angle X-ray scattering: insights from an extended examination of selected data in the Small-Angle Scattering Biological Data Bank.

By providing predicted protein structures from nearly all known protein sequences, the artificial intelligence program AlphaFold (AF) is having a major impact on structural biology. While a stunning accuracy has been achieved for many folding units, predicted unstructured regions and the arrangement of potentially flexible linkers connecting structured domains present challenges. Focusing on single-chain structures without prosthetic groups, an earlier comparison of features derived from small-angle X-ray scattering (SAXS) data taken from the Small-Angle Scattering Biological Data Bank (SASBDB) is extended to those calculated using the corresponding AF-predicted structures. Selected SASBDB entries were carefully examined to ensure that they represented data from monodisperse protein solutions and had sufficient statistical precision and q resolution for reliable structural evaluation. Three examples were identified where there is clear evidence that the single AF-predicted structure cannot account for the experimental SAXS data. Instead, excellent agreement is found with ensemble models generated by allowing for flexible linkers between high-confidence predicted structured domains. A pool of representative structures was generated using a Monte Carlo method that adjusts backbone dihedral allowed angles along potentially flexible regions. A fast ensemble modelling method was employed that optimizes the fit of pair distance distribution functions [P(r) versus r] and intensity profiles [I(q) versus q] computed from the pool to their experimental counterparts. These results highlight the complementarity between AF prediction, solution SAXS and molecular dynamics/conformational sampling for structural modelling of proteins having both structured and flexible regions.

A functional unit combination strategy for enhancing red room-temperature phosphorescence.

Red room-temperature phosphorescence (RTP) materials based on non-metallic organic compounds are less reported compared to the commonly found green RTP materials. Here, we propose a novel approach to obtain red RTP materials by integrating and combining two functional units, resembling a jigsaw puzzle. In this approach, benzo[c][2,1,3]thiadiazole (BZT) serves as the red RTP unit, while a folding unit containing sulphur/oxygen is responsible for enhancing spin-orbit coupling (SOC) to accelerate the intersystem crossing (ISC) process. Three new molecules (SS-BZT, SO-BZT, and OO-BZT) were designed and synthesized, among which SS-BZT and SO-BZT with folded geometries demonstrate enhanced red RTP in their monodisperse films compared to the parent BZT. Meanwhile, the SS-BZT film shows a dual emission consisting of blue fluorescence and red RTP, with a significant spectral separation of approximately 150 nm, which makes the SS-BZT film highly suitable for applications in optical oxygen sensing and ratiometric detection. Within the oxygen concentration range of 0-1.31%, the SS-BZT film demonstrates a quenching constant of 2.66 kPa-1 and a quenching efficiency of 94.24%, indicating that this probe has the potential to accurately detect oxygen in a hypoxic environment.

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction.

Most proteins in cells are composed of multiple folding units (or domains) to perform complex functions in a cooperative manner. Relative to the rapid progress in single-domain structure prediction, there are few effective tools available for multi-domain protein structure assembly, mainly due to the complexity of modeling multi-domain proteins, which involves higher degrees of freedom in domain-orientation space and various levels of continuous and discontinuous domain assembly and linker refinement. To meet the challenge and the high demand of the community, we developed I-TASSER-MTD to model the structures and functions of multi-domain proteins through a progressive protocol that combines sequence-based domain parsing, single-domain structure folding, inter-domain structure assembly and structure-based function annotation in a fully automated pipeline. Advanced deep-learning models have been incorporated into each of the steps to enhance both the domain modeling and inter-domain assembly accuracy. The protocol allows for the incorporation of experimental cross-linking data and cryo-electron microscopy density maps to guide the multi-domain structure assembly simulations. I-TASSER-MTD is built on I-TASSER but substantially extends its ability and accuracy in modeling large multi-domain protein structures and provides meaningful functional insights for the targets at both the domain- and full-chain levels from the amino acid sequence alone.

DEMO2: Assemble multi-domain protein structures by coupling analogous template alignments with deep-learning inter-domain restraint prediction.

Most proteins in nature contain multiple folding units (or domains). The revolutionary success of AlphaFold2 in single-domain structure prediction showed potential to extend deep-learning techniques for multi-domain structure modeling. This work presents a significantly improved method, DEMO2, which integrates analogous template structural alignments with deep-learning techniques for high-accuracy domain structure assembly. Starting from individual domain models, inter-domain spatial restraints are first predicted with deep residual convolutional networks, where full-length structure models are assembled using L-BFGS simulations under the guidance of a hybrid energy function combining deep-learning restraints and analogous multi-domain template alignments searched from the PDB. The output of DEMO2 contains deep-learning inter-domain restraints, top-ranked multi-domain structure templates, and up to five full-length structure models. DEMO2 was tested on a large-scale benchmark and the blind CASP14 experiment, where DEMO2 was shown to significantly outperform its predecessor and the state-of-the-art protein structure prediction methods. By integrating with new deep-learning techniques, DEMO2 should help fill the rapidly increasing gap between the improved ability of tertiary structure determination and the high demand for the high-quality multi-domain protein structures. The DEMO2 server is available at https://zhanggroup.org/DEMO/.

Network Representation Learning Algorithm Based on Community Folding

<p>Network representation learning is a machine learning method that maps network topology and node information into low-dimensional vector space, which can reduce the temporal and spatial complexity of downstream network data mining such as node classification and graph clustering. This paper addresses the problem that neighborhood information-based network representation learning algorithm ignores the global topological information of the network. We propose the Network Representation Learning Algorithm Based on Community Folding (CF-NRL) considering the influence of community structure on the global topology of the network. Each community of the target network is regarded as a folding unit, the same network representation learning algorithm is used to learn the vector representation of the nodes on the folding network and the target network, then the vector representations are spliced correspondingly to obtain the final vector representation of the node. Experimental results show the excellent performance of the proposed algorithm.</p> <p> </p>

METHOD OF "SKELETAL STRUCTURES" TO SIMPLIFY THE PARAMETRIZATION PROCESS IN AUTOCAD

The article considers the method of "skeletal structures", proposed to simplify the process of parameterization. When parameterizing objects whose shape has more than one contour, there may be some complications, namely: auto-overlay dependencies, or sequential parameterization of the relative position of image fragments, can lead to an excessive number of dependencies, which can lead to inconsistencies when performing parameterization . The proposed method allows you to minimize such inconveniences, reduce the number of superimposed parameters and improve the quality of control over the shape of the object. The stages of parameterization of objects by the method of "skeletal structures" are formulated and characterized and recommendations for its use are given.
 The method of "skeletal structures" was developed and applied in the process of studying the topic "Parameterization" of the course "Engineering and Computer Graphics" at the Department of Descriptive Geometry, Engineering and Computer Graphics of the National Technical University of Ukraine "Kyiv Polytechnic Institute named after Igor Sikorsky". The application of the method on the example of flat parts of complex shape and for the structures used in the study of the topic "Assembly drawing" is demonstrated. As a condition of the task in the topic "Assembly drawing" we used a parameterized folding unit, which students adjusted to the parameters corresponding to the variant of the task, and supplemented the drawing with simplified images of fastening threaded elements made by them in the form of dynamic blocks.

Dissecting Inter-domain Cooperativity in the Folding of a Multi Domain Protein

Correct protein folding underlies all cellular functions. While there are detailed descriptions and a good understanding of protein folding pathways for single globular domains there is a paucity of quantitative data regarding folding of multidomain proteins. We have here investigated the folding of a three-domain supramodule from the protein PSD-95, consisting of one PDZ domain, one SH3 domain and one guanylate kinase-like (GK) domain. This supramodule has previously been shown to work as one functional unit with regard to ligand binding. We used equilibrium and kinetic folding experiments to demonstrate that the PDZ domain folds faster and independently from the SH3-GK tandem, which folds as one cooperative unit. However, concurrent folding of the PDZ domain slows down folding of SH3-GK by non-native interactions, resulting in an off-pathway folding intermediate. Our data contribute to an emerging description of multidomain protein folding in which individual domains cannot a priori be viewed as separate folding units.

Using Folding Structure to Enhance Measurement Range, Sensitivity of the Flexible Sensors: A Simple, Eco‐Friendly, and Effective Method

AbstractFlexible sensing technology has shown great application value in motion monitoring and physiological signal detection. In many applications, the detected signal has the characteristics of low strength and wide range, thus improving the sensitivity and measurement range of sensors becomes extremely important. An enhancing method based on folding structure is proposed in this paper. Through the analysis of folding structure characteristics, the theoretical model of the folding structure effect in flexible sensors is established and the enhancement effect of sensitivity and measurement range is testified through three kinds of commonly used sensors, resistive, capacitive, and inductive sensors. Through changing the number of folding units, the sensitivity and measurement range of sensors can achieve to be adjusted. Folding sensors also have good hysteresis and repeatability properties and show the great performance in the application of human motion monitoring, such as limbs bending and rotating, muscle powering. The proposed method has the characteristics of simple, economical, environment friendly, and widely suitable, meanwhile avoiding the complexity of other ways to improve performance. This work has potential to enhance the sensitivity and measurement range for more kinds of flexible sensors and is of great value to fabricate high performance sensors based on folding structure.

Design of landing of assembly machine building units with circulating load rolling bearing rings

The article presents the results of solving the scientific problem of ensuring folding, which is complex and includes the appointment of optimal tolerances, maximum deviations and landings in the design. The authors presented for selection of locally loaded bearing rings the field of tolerances of holes and shafts depending on type of bearings, operating conditions and the nature of load. The authors consider the definition of the dynamic coefficient through the value of the landing on the minimum tension between the circulating load ring and the surface of the connected part. Checking the correctness of the choice of landing involves ensuring the condition of strength at the allowable tension. The authors determined the equivalent dynamic load for the established requirements for design of rolling bearings, taking into account the load variability of 6005 N and calculated the durability of 9500 hours. In article it is established that on diameters of openings of demountable cases under external rings with local loading it is expedient to assign fields of tolerances H6, H7, G6, G7. The article confirms the possibility of periodic rotation of the bearing ring during the operation of the folding unit and reduce the uneven wear of the raceways.

Domain Interactions Determine the Conformational Ensemble of the Periplasmic Chaperone Sura

SurA is thought to be the most important periplasmic chaperone for outer membrane protein (OMP) biogenesis. Its structure is composed of a core region and two peptidylprolyl isomerase domains, termed P1 and P2, connected by flexible linkers. As such these three independent folding units are able to adopt a number of distinct spatial positions with respect to each other. The conformational dynamics of these domains are thought to be functionally important yet are largely unresolved. Here we address this question of the conformational ensemble using sedimentation equilibrium, small-angle neutron scattering, and folding titrations. This combination of orthogonal methods converges on a SurA population that is monomeric at physiological concentrations. The conformation that dominates this population has the P1 and core domains docked to one another, for example, "P1-closed" and the P2 domain extended in solution. We discovered that the distribution of domain orientations is defined by modest and favorable interactions between the core domain and either the P1 or the P2 domains. These two peptidylprolyl domains compete with each other for core-binding but are thermodynamically uncoupled. This arrangement implies two novel insights. Firstly, an open conformation must exist to facilitate P1 and P2 exchange on the core, indicating that the open client-binding conformation is populated at low levels even in the absence of client unfolded OMPs. Secondly, competition between P1 and P2 binding paradoxically occludes the client binding site on the core, which may serve to preserve the reservoir of binding-competent apo-SurA in the periplasm.

Programmed Trade-offs in Protein Folding Networks

Molecular chaperones as specialized protein quality control enzymes form the core of cellular protein homeostasis. How chaperones selectively interact with their substrate proteins thus allocate their overall limited capacity remains poorly understood. Here, I present an integrated analysis of sequence and structural determinants that define interactions of protein domains as the basic protein folding unit with the Saccharomyces cerevisiae Hsp70 Ssb. Structural homologs of single-domain proteins that differentially interact with Ssb for de novo folding were found to systematically differ in complexity of their folding landscapes, selective use of nonoptimal codons, and presence of short discriminative sequences, thus highlighting pervasive trade-offs in chaperone-assisted protein folding landscapes. However, short discriminative sequences were found to contribute by far the strongest signal toward explaining Ssb interactions. This observation suggested that some chaperone interactions may be directly programmed in the amino acid sequences rather than responding to folding challenges, possibly for regulatory advantages.