Protein Quaternary Structure Research Articles

Martini 3 OliGo̅mers: A Scalable Approach for Multimers and Fibrils in GROMACS.

Martini 3 is a widely used coarse-grained simulation method for large-scale biomolecular simulations. It can be combined with a Go̅ model to realistically describe higher-order protein structures while allowing the folding and unfolding events. However, as of today, this method has largely been used only for individual monomers. In this article, we describe how the Go̅ model can be implemented within the framework of Martini 3 for a multimer system, taking into account both intramolecular and intermolecular interactions in an oligomeric protein system. We demonstrate the method by showing how it can be applied to both structural stability maintenance and assembly/disassembly of protein oligomers, using aquaporin tetramer, insulin dimer, and amyloid-β fibril as examples. We find that addition of intermolecular Go̅ potentials stabilizes the quaternary structure of proteins. The strength of the Go̅ potentials can be tuned so that the internal fluctuations of proteins match the behavior of atomistic simulation models, however, the results also show that the use of too strong intermolecular Go̅ potentials weakens the chemical specificity of oligomerization. The Martini-Go̅ model presented here enables the use of Go̅ potentials in oligomeric molecular systems in a computationally efficient and parallelizable manner, especially in the case of homopolymers, where the number of identical protein monomers is high. This paves the way for coarse-grained simulations of large protein complexes, such as viral protein capsids and prion fibrils, in complex biological environments.

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.

IMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.

Performance and protein conformation of thermally treated silver carp (Hypophthalmichthys molitrix) and scallop (Argopecten irradians) blended gels.

The quality of surimi-based products can be improved by combining the flesh of different aquatic organisms. The present study investigated the effects of incorporating diverse ratios of unwashed silver carp (H) and scallop (A) and using various thermal treatments on the moisture, texture, microstructure, and conformation of the blended gels and myofibrillar protein of surimi. A mixture ratio of A:H = 1:3 yielded the highest gel strength, which was 60.4% higher than that of scallop gel. The cooking losses of high-pressure heating and water-bath microwaving were significantly higher than those of other methods (P < 0.05). Moreover, the two-step water bath and water-bath microwaving samples exhibited a more regular spatial network structure compared to other samples. The mixed samples exhibited a microstructure with a uniform and ordered spatial network, allowing more free water to be trapped by the internal structure, resulting in more favorable gel properties. The thermal treatments comprehensively modified the tertiary and quaternary structures of proteins in unwashed mixed gel promoted protein unfurling, provided more hydrophobic interactions, enhanced protein aggregation and improved the gel performance. The findings of the present study improve our understanding of the interactions between proteins from different sources. We propose a new method for modifying surimi's gel properties, facilitating the development of mixed surimi products, as well as enhancing the efficient utilization of aquatic resources. © 2024 Society of Chemical Industry.

Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

A Survey of Deep Learning Methods for Estimating the Accuracy of Protein Quaternary Structure Models.

The quality prediction of quaternary structure models of a protein complex, in the absence of its true structure, is known as the Estimation of Model Accuracy (EMA). EMA is useful for ranking predicted protein complex structures and using them appropriately in biomedical research, such as protein-protein interaction studies, protein design, and drug discovery. With the advent of more accurate protein complex (multimer) prediction tools, such as AlphaFold2-Multimer and ESMFold, the estimation of the accuracy of protein complex structures has attracted increasing attention. Many deep learning methods have been developed to tackle this problem; however, there is a noticeable absence of a comprehensive overview of these methods to facilitate future development. Addressing this gap, we present a review of deep learning EMA methods for protein complex structures developed in the past several years, analyzing their methodologies, data and feature construction. We also provide a prospective summary of some potential new developments for further improving the accuracy of the EMA methods.

Cryoprotective effect of tamarind seed polysaccharide on grass carp surimi: characteristics, interactions, and mechanisms

In surimi products, the quality deterioration caused by freezing is a common problem. Therefore, the development of natural cryoprotectants is receiving increased attention. Tamarind seed polysaccharide (TSP) has been found to have a superior ice recrystallization inhibitory effect. In this work, the cryoprotective effect of TSP on grass carp surimi was investigated, including freeze-thaw stability, water distribution, texture, and microstructure. Additionally, the interaction between TSP and myofibrillar protein was analyzed. The inclusion of TSP resulted in the formation of a more regular and compact gel network through hydrogen bonding, which enhanced the binding between water molecules and myofibrillar protein. This, in turn, immobilized free water within the gel network. Furthermore, TSP prevented the texture deterioration of the surimi gel during repeated freeze-thaw cycles, particularly when 1.0% TSP was added. Moreover, the presence of TSP delayed the decline of Ca2+-ATPase activity and stabilized the secondary, tertiary, and quaternary structure of myofibrillar protein during freeze-thaw cycles. Therefore, TSP shows promise as a potential cryoprotectant for frozen surimi products, enhancing their quality and stability during frozen storage.

Mutational biases favor complexity increases in protein interaction networks after gene duplication

Biological systems can gain complexity over time. While some of these transitions are likely driven by natural selection, the extent to which they occur without providing an adaptive benefit is unknown. At the molecular level, one example is heteromeric complexes replacing homomeric ones following gene duplication. Here, we build a biophysical model and simulate the evolution of homodimers and heterodimers following gene duplication using distributions of mutational effects inferred from available protein structures. We keep the specific activity of each dimer identical, so their concentrations drift neutrally without new functions. We show that for more than 60% of tested dimer structures, the relative concentration of the heteromer increases over time due to mutational biases that favor the heterodimer. However, allowing mutational effects on synthesis rates and differences in the specific activity of homo- and heterodimers can limit or reverse the observed bias toward heterodimers. Our results show that the accumulation of more complex protein quaternary structures is likely under neutral evolution, and that natural selection would be needed to reverse this tendency.

Polymorphic Self-Assembly with Procedural Flexibility for Monodisperse Quaternary Protein Structures of DegQ Enzymes.

As large molecular tertiary structures, some proteins can act as small robots that find, bind, and chaperone target protein clients, showing the potential to serve as smart building blocks in self-assembly fields. Instead of using such intrinsic functions, most self-assembly methodologies for proteins aim for de novo-designed structures with accurate geometric assemblies, which can limit procedural flexibility. Here, a strategy enabling polymorphic clustering of quaternary proteins, exhibiting simplicity and flexibility of self-assembling paths for proteins in forming monodisperse quaternary cage particles is presented. It is proposed that the enzyme protomer DegQ, previously solved at low resolution, may potentially be usable as a threefold symmetric building block, which can form polyhedral cages incorporated by the chaperone action of DegQ in the presence of protein clients. To obtain highly monodisperse cage particles, soft, and hence, less resistive client proteins, which can program the inherent chaperone activity of DegQ to efficient formations of polymorphic cages, depending on the size of clients are utilized. By reconstructing the atomic resolution cryogenic electron microscopy DegQ structures using obtained 12- and 24-meric clusters, the polymorphic clustering of DegQ enzymes is validated in terms of soft and rigid domains, which will provide effective routes for protein self-assemblies with procedural flexibility.

Creation of a point-of-care therapeutics sensor using protein engineering, electrochemical sensing and electronic integration

Point-of-care sensors, which are low-cost and user-friendly, play a crucial role in precision medicine by providing quick results for individuals. Here, we transform the conventional glucometer into a 4-hydroxytamoxifen therapeutic biosensor in which 4-hydroxytamoxifen modulates the electrical signal generated by glucose oxidation. To encode the 4-hydroxytamoxifen signal within glucose oxidation, we introduce the ligand-binding domain of estrogen receptor-alpha into pyrroloquinoline quinone-dependent glucose dehydrogenase by constructing and screening a comprehensive protein insertion library. In addition to obtaining 4-hydroxytamoxifen regulatable engineered proteins, these results unveil the significance of both secondary and quaternary protein structures in propagation of conformational signals. By constructing an effective bioelectrochemical interface, we detect 4-hydroxytamoxifen in human blood samples as changes in the electrical signal and use this to develop an electrochemical algorithm to decode the 4-hydroxytamoxifen signal from glucose. To meet the miniaturization and signal amplification requirements for point-of-care use, we harness power from glucose oxidation to create a self-powered sensor. We also amplify the 4-hydroxytamoxifen signal using an organic electrochemical transistor, resulting in milliampere-level signals. Our work demonstrates a broad interdisciplinary approach to create a biosensor that capitalizes on recent innovations in protein engineering, electrochemical sensing, and electrical engineering.

Comparative physicochemical and structural characterisation studies establish high biosimilarity between BGL-ASP and reference insulin aspart

Biosimilar insulin analogues are increasing market access for diabetic patients globally. Scientific establishment of biosimilarity is cornerstone of this key change in the medical landscape. BGL-ASP is a biosimilar insulin aspart developed by BioGenomics Limited, India. BioGenomics has considered a stepwise approach in generating the totality of evidence required to establish similarity with reference product. Insulin aspart is a recombinant rapid-acting human insulin analogue utilised in the treatment of type-1 and type-2 diabetes mellitus. The single amino acid substitution at position B28 where proline is replaced with aspartic acid results in a decreased propensity to form hexamers, thus increasing the absorption rate on subcutaneous administration compared to native insulin. In order to establish the safety and efficacy of BGL-ASP, the critical quality attributes (CQAs) of BGL-ASP are identified based on the impact created on biological activity, pharmacokinetic/pharmacodynamic (PK/PD), immunogenicity and safety. The CQAs of insulin aspart are related to product structure, purity and functionality and are characterised using a series of state-of-the-art orthogonal analytical tools. The primary protein sequence, the secondary, tertiary and quaternary structure are found to be highly similar for BGL-ASP and reference product. The product related impurities of insulin aspart and the assay content are determined using high performance liquid chromatography (HPLC) based analysis and is similar for BGL-ASP and reference insulin aspart sourced from United States of America (US), Europe Union (EU) and India. The safety, efficacy and immunogenicity of BGL-ASP is also found to be comparable with reference product and is confirmed through the clinical trials conducted as recommended by International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH) and European Medicines Agency (EMA) guidelines. The data encompassed in this study demonstrates that reference insulin aspart and BGL-ASP are highly similar in terms of structural, physicochemical, and biological properties, thus confirming its safety and efficacy for usage as potential alternative economical medicinal treatment for diabetes mellitus.

Functional implications of unusual NOS and SONOS covalent linkages found in proteins.

The tertiary and quaternary structures of many proteins are stabilized by strong covalent forces, of which disulfide bonds are the most well known. A new type of intramolecular and intermolecular covalent bond has been recently reported, consisting of the Lys and Cys side-chains linked by an oxygen atom (NOS). These post-translational modifications are widely distributed amongst proteins, and are formed under oxidative conditions. Similar linkages are observed during antibiotic biosynthesis, where hydroxylamine intermediates are tethered to the sulfur of enzyme active site Cys residues. These linkages open the way to understanding protein structure and function, give new insights into enzyme catalysis and natural product biosynthesis, and offer new strategies for drug design.

Electrostatic origin of a stabilizing synergistic interaction among b-, c-, and f-residues in a trimeric coiled coil.

Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation. Whereas the roles of residues at the canonical heptad positions the a, d, e, and g are understood in precise detail, conventional approaches often assume that the solvent-exposed b-, c-, and f-positions can be varied broadly for application-specific purposes with minimal consequences. However, a growing body of evidence suggests that interactions among these b, c, and f residues can contribute substantially to coiled-coil conformational stability. In the trimeric coiled coil described here, we find that b-position Glu10 engages in a stabilizing long-range synergistic interaction with c-position Lys18 (ΔΔΔGf = -0.65 ± 0.02 kcal/mol). This favorable interaction depends strongly on the presence of two nearby f-position residues: Lys 7 and Tyr14. Extensive mutational analysis of these residues in the presence of added salt vs. denaturant suggests that this long-range synergistic interaction is primarily electrostatic in origin, but also depends on the precise location and acidity of a side-chain hydrogen-bond donor within f-position Tyr14.

Self-Assembled Protein Nanostructures via Irreversible Peptide Assembly.

The quaternary structure of proteins extends the functionality of monomeric proteins. Similarly, self-assembled protein nanostructures (SPrNs) have great potential to improve the functionality and complexity of proteins; however, the difficulty associated with the fabrication of SPrNs is far greater than that associated with the fabrication of self-assembled peptides or polymers and often requires sophisticated computational design. To make the process of SPrN formation simpler and more intuitive, herein, we devise a strategy to adopt an irreversible self-assembled peptide nanostructure (SPeN) process en route to the formation of SPrNs. The strategy employs three sequential steps: first, the formation of SPeNs (an equilibrium process); second, covalent capture of SPeNs (an irreversible process); third, the final assembly of SPrNs via protein-peptide interactions (an equilibrium process). This strategy allowed us to fabricate SPrNs in which the size of the protein was approximately 9 times higher than that of the self-assembling peptide. Furthermore, we demonstrated that the irreversible SPeN could be used as a primary building block for assembly into superstructures. Overall, this strategy is conceptually as simple as SPeN fabrication and is potentially applicable to any soluble protein.

Development of Pan-Anti-SARS-CoV-2 Agents through Allosteric Inhibition of nsp14/nsp10 Complex.

SARS-CoV-2 nsp14 functions both as an exoribonuclease (ExoN) together with its critical cofactor nsp10 and as an S-adenosyl methionine-dependent (guanine-N7) methyltransferase (MTase), which makes it an attractive target for the development of pan-anti-SARS-CoV-2 drugs. Herein, we screened a panel of compounds (and drugs) and found that certain compounds, especially Bi(III)-based compounds, could allosterically inhibit both MTase and ExoN activities of nsp14 potently. We further demonstrated that Bi(III) binds to both nsp14 and nsp10, resulting in the release of Zn(II) ions from the enzymes as well as alternation of protein quaternary structures. The in vitro activities of the compounds were also validated in SARS-CoV-2-infected mammalian cells. Importantly, we showed that nsp14 serves as an authentic target of Bi(III)-based antivirals in SARS-CoV-2-infected mammalian cells by quantification of both the protein and inhibitor. This study highlights the importance of nsp14/nsp10 as a potential target for the development of pan-antivirals against SARS-CoV-2 infection.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Structural Expansion of Catalytic RNA Nanostructures through Oligomerization of a Cyclic Trimer of Engineered Ribozymes.

The multimolecular assembly of three-dimensionally structured proteins forms their quaternary structures, some of which have high geometric symmetry. The size and complexity of protein quaternary structures often increase in a hierarchical manner, with simpler, smaller structures serving as units for larger quaternary structures. In this study, we exploited oligomerization of a ribozyme cyclic trimer to achieve larger ribozyme-based RNA assembly. By installing kissing loop (KL) interacting units to one-, two-, or three-unit RNA molecules in the ribozyme trimer, we constructed dimers, open-chain oligomers, and branched oligomers of ribozyme trimer units. One type of open-chain oligomer preferentially formed a closed tetramer containing 12 component RNAs to provide 12 ribozyme units. We also observed large assembly of ribozyme trimers, which reached 1000 nm in size.

Precision Macro (molecular) Assemblies

Structural precision in hierarchical self-assembled architectures is ubiquitous in biological systems. For instance, DNA double helix, quaternary structures of proteins, tobacco mosaic virus, microtubules, and several other elegant systems are constituted through specific molecular recognitions that are responsible for regulating numerous complex functions in nature. Inspired by such elegant examples in living systems, the self-assembly of synthetic small molecules and macromolecules has been studied with great interest, especially in the past four decades since the concept of supramolecular chemistry had been established in a formal way. In this context, solution self-assembly of amphiphilic block copolymers (ABCPs) with amorphous core- forming blocks has been studied extensively in the past three decades owing to the possibility of generating different nanostructures in water,such as micelles, vesicles, fibers, lamella, and others.Despite the fact that self-assembly of ABCPs has proven to be an effective bottom- up approach for creating multilevel structures that are gateways to highly promising soft materials for diverse applications, attempts to improve specific and nontrivial functional utilities of these systems have been hampered by a number of significant problems. Aggregation of ABCPs in block-selective solvents is commonly catalyzed by the immiscibility-driven chain collapse of the amorphous core-forming blocks. Thus, the critical packing parameter, which in turn depends on the relative volume fraction of the hydrophobic and hydrophilic block segments, is the primary factor controlling the morphology of these polymer aggregates in solutions.This limits the ability to precisely tailor the internal order orsurface functional group display in such nanostructures or control their dimensions, shapes, and dispersity, which is crucial for their elegant biological functions. Moreover, for ABCPs with amorphous core- forming blocks, non-spherical structures are rarely accessible only within a limited window of block compositions and self-assembly environments. Sometimes, those are also contaminated with other undesired morphologies. Many studies have demonstrated that non-spherical, high aspect ratio nanostructures such as one- dimensional (1D) fibers and two- dimensional (2D) plateletperform superior functions in emerging applications, compared to their spherical analogues. This has paved the way for the emergence of new macromolecular designs toward the development of internally-ordered precision anisotropic structures.

β-Sheet Assembly Translates Conservative Single-Site Mutation into a Perturbation in Macroscopic Structure.

Transferring structural information from amino acid sequence to macroscale assembly is a challenging approach for designing protein quaternary structure. However, the pathway by which the slight variations in sequence result in a global perturbation effect on the assembled structure is unknown. Herein, we design two synthetic peptides, QNL-His and QNL-Arg, with one amino acid substitution and use scanning tunneling microscopy (STM) to image individual peptides in the assembled state. The submolecular resolution of STM enables us to determine the folding structure and β-sheet supramolecular organization of peptides. QNL-His and QNL-Arg differ in their β-strand length distribution in pleated β-sheet association. These structural variations lead to distinguishable outcomes in their β-sheet assembled fibrils and phase transitions. The comparison of QNL-His versus QNL-Arg structures and macroscopic properties unveils the role of assembly to amplify the structural variations associated with a single-site mutation from a single-molecule scale to a macroscopic scale.