DeepSS2GO: protein function prediction from secondary structure.

Predicting protein function is crucial for understanding biological life processes, preventing diseases and developing new drug targets. In recent years, methods based on sequence, structure and biological networks for protein function annotation have been extensively researched. Although obtaining a protein in three-dimensional structure through experimental or computational methods enhances the accuracy of function prediction, the sheer volume of proteins sequenced by high-throughput technologies presents a significant challenge. To address this issue, we introduce a deep neural network model DeepSS2GO (Secondary Structure to Gene Ontology). It is a predictor incorporating secondary structure features along with primary sequence and homology information. The algorithm expertly combines the speed of sequence-based information with the accuracy of structure-based features while streamlining the redundant data in primary sequences and bypassing the time-consuming challenges of tertiary structure analysis. The results show that the prediction performance surpasses state-of-the-art algorithms. It has the ability to predict key functions by effectively utilizing secondary structure information, rather than broadly predicting general Gene Ontology terms. Additionally, DeepSS2GO predicts five times faster than advanced algorithms, making it highly applicable to massive sequencing data. The source code and trained models are available at https://github.com/orca233/DeepSS2GO.

Multiscale Simulations of Self-Assembling Peptides: Surface and Core Hydrophobicity Determine Fibril Stability and Amyloid Aggregation.

Assemblies of peptides and proteins through specific intermolecular interactions set the basis for macroscopic materials found in nature. Peptides provide easily tunable hydrogen-bonding interactions, which can lead to the formation of ordered structures such as highly stable ß-sheets that can form amyloid-like supramolecular peptide nanofibrils (PNFs). PNFs are of special interest, as they could be considered as mimics of various fibrillar structures found in nature. In their ability to serve as supramolecular scaffolds, they could mimic certain features of the extracellular matrix to provide stability, interact with pathogens such as virions, and transduce signals between the outside and inside of cells. Many PNFs have been reported that reveal rich bioactivities. PNFs supporting neuronal cell growth or lentiviral gene transduction have been studied systematically, and their material properties were correlated to bioactivities. However, the impact of the structure of PNFs, their dynamics, and stabilities on their unique functions is still elusive. Herein, we provide a microscopic view of the self-assembled PNFs to unravel how the amino acid sequence of self-assembling peptides affects their secondary structure and dynamic properties of the peptides within supramolecular fibrils. Based on sequence truncation, amino acid substitution, and sequence reordering, we demonstrate that peptide-peptide aggregation propensity is critical to form bioactive ß-sheet-rich structures. In contrast to previous studies, a very high peptide aggregation propensity reduces bioactivity due to intermolecular misalignment and instabilities that emerge when fibrils are in close proximity to other fibrils in solution. Our multiscale simulation approach correlates changes in biological activity back to single amino acid modifications. Understanding these relationships could lead to future material discoveries where the molecular sequence predictably determines the macroscopic properties and biological activity. In addition, our studies may provide new insights into naturally occurring amyloid fibrils in neurodegenerative diseases.

Toward Synthetic Intrinsically Disordered Polypeptides (IDPs): Controlled Incorporation of Glycine in the Ring-Opening Polymerization of N-Carboxyanhydrides.

Intrinsically disordered proteins (IDPs) do not have a well-defined folded structure but instead behave as extended polymer chains in solution. Many IDPs are rich in glycine residues, which create steric barriers to secondary structuring and protein folding. Inspired by this feature, we have studied how the introduction of glycine residues influences the secondary structure of a model polypeptide, poly(l-glutamic acid), a helical polymer. For this purpose, we carried out ring-opening copolymerization with γ-benzyl-l-glutamate and glycine N-carboxyanhydride (NCA) monomers. We aimed to control the glycine distribution within PBLG by adjusting the reactivity ratios of the two NCAs using different reaction conditions (temperature, solvent). The relationship between those conditions, the monomer distributions, and the secondary structure enabled the design of intrinsically disordered polypeptides when a highly gradient microstructure was achieved in DMSO.

DSSP in GROMACS: Tool for Defining Secondary Structures of Proteins in Trajectories.

This work describes a fast implementation of a software algorithm associated with determination of protein secondary structure based on the Define Secondary Structure of Proteins (DSSP) algorithm. This implementation is fully compatible with the DSSP v.4 and DSSP v.2 algorithms and implemented as a native GROMACS trajectory analysis module, which allows us to analyze molecular dynamics trajectories without any restrictions of the original DSSP implementation. This implementation works much faster than the original DSSP v.4 and DSSP v.2 algorithms.

Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties.

Contaminating microplastics can interact with food proteins in the food matrix and during digestion. This study investigated adsorption of chicken egg protein ovalbumin to polystyrene (PS, 110 and 260 µm) and polyethylene terephthalate (PET, 140 µm) MPs in acidic and neutral conditions and alterations in ovalbumin structure. Ovalbumin adsorption affinity depended on MPs size (smaller > larger), type (PS > PET) and pH (pH 3 > pH 7). In bulk solution, MPs does not change ovalbumin secondary structure significantly, but induces loosening (at pH 3) and tightening (at pH 7) of tertiary structure. Formed soft corona exclusively consists of full length non-native ovalbumin, while in hard corona also shorter ovalbumin fragments were found. At pH 7 soft corona ovalbumin has rearranged but still preserved level of ordered secondary structure, resulting in preserved thermostability and proteolytic stability, but decreased ability to form fibrils upon heating. Secondary structure changes in soft corona resemble changes in native ovalbumin induced by heat treatment (80 °C). Ovalbumin is abundantly present in corona around microplastics also in the presence of other egg white proteins. These results imply that microplastics contaminating food may bind and change structure and functional properties of the main egg white protein.

MFTrans: A multi-feature transformer network for protein secondary structure prediction.

In the rapidly evolving field of computational biology, accurate prediction of protein secondary structures is crucial for understanding protein functions, facilitating drug discovery, and advancing disease diagnostics. In this paper, we propose MFTrans, a deep learning-based multi-feature fusion network aimed at enhancing the precision and efficiency of Protein Secondary Structure Prediction (PSSP). This model employs a Multiple Sequence Alignment (MSA) Transformer in combination with a multi-view deep learning architecture to effectively capture both global and local features of protein sequences. MFTrans integrates diverse features generated by protein sequences, including MSA, sequence information, evolutionary information, and hidden state information, using a multi-feature fusion strategy. The MSA Transformer is utilized to interleave row and column attention across the input MSA, while a Transformer encoder and decoder are introduced to enhance the extracted high-level features. A hybrid network architecture, combining a convolutional neural network with a bidirectional Gated Recurrent Unit (BiGRU) network, is used to further extract high-level features after feature fusion. In independent tests, our experimental results show that MFTrans has superior generalization ability, outperforming other state-of-the-art PSSP models by 3 % on average on public benchmarks including CASP12, CASP13, CASP14, TEST2016, TEST2018, and CB513. Case studies further highlight its advanced performance in predicting mutation sites. MFTrans contributes significantly to the protein science field, opening new avenues for drug discovery, disease diagnosis, and protein.

Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability.

Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═Oi···Hi+4-N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both i/i + 4 hydrogen bonds and i/i + 1 n → π* interactions. This hypothesis was tested in peptides X-AKAAAAKAAAAKAAGY-NH2, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and iso-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water-amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide-water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.

Solution structure and pressure response of thioredoxin-1 of Plasmodium falciparum.

We present here the solution structures of the protein thioredoxin-1 from Plasmodium falciparum (PfTrx-1), in its reduced and oxidized forms. They were determined by high-resolution NMR spectroscopy at 293 K on uniformly 13C-, 15N-enriched, matched samples allowing to identification of even small structural differences. PfTrx-1 shows an α/ß-fold with a mixed five-stranded ß-sheet that is sandwiched between 4 helices in a ß1 α1 ß2 α2 ß3 α3 ß4 ß5 α4 topology. The redox process of the CGPC motif leads to significant structural changes accompanied by larger chemical shift changes from residue Phe25 to Ile36, Thr70 to Thr74, and Leu88 to Asn91. By high-field high-pressure NMR spectroscopy, rare conformational states can be identified that potentially are functionally important and can be used for targeted drug development. We performed these experiments in the pressure range from 0.1 MPa to 200 MPa. The mean combined, random-coil corrected B1* values of reduced and oxidized thioredoxin are quite similar with -0.145 and -0.114 ppm GPa-1, respectively. The mean combined, random-coil corrected B2* values in the reduced and oxidized form are 0.179 and 0.119 ppm GPa-2, respectively. The mean ratios of the pressure coefficients B2/B1 are -0.484 and -0.831 GPa-1 in the reduced and oxidized form respectively. They differ at some points in the structure after the formation of the disulfide bond between C30 and C33. The thermodynamical description of the pressure dependence of chemical shifts requires the assumption of at least three coexisting conformational states of PfTrx-1. These three conformational states were identified in the reduced as well as in the oxidized form of the protein, therefore, they represent sub-states of the two main oxidation states of PfTrx-1.

Dual-Capped Helical Interface Mimics.

Disruption of protein-protein interactions is medicinally important. Interface helices may be mimicked in helical probes featuring enhanced rigidities, binding to protein targets, stabilities in serum, and cell uptake. This form of mimicry is dominated by stapling between side chains of helical residues: there has been less progress on helical N-caps, and there were no generalizable C-caps. Conversely, in natural proteins, helicities are stabilized and terminated by C- and N-caps but not staples. Bicyclic caps previously introduced by us enable interface helical mimicry featuring rigid synthetic caps at both termini in this work. An unambiguously helical dual-capped system proved to be conformationally stable, binding cyclins A and E, and showed impressive cellular uptake. In addition, the dual-capped mimic was completely resistant to proteolysis in serum over an extended period when compared with "gold standard" hydrocarbon-stapled controls. Dual-capped peptidomimetics are a new, generalizable paradigm for helical interface probe design.

Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX.

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

High-throughput Selection of Human de novo-emerged sORFs with High Folding Potential.

De novo genes emerge from previously noncoding stretches of the genome. Their encoded de novo proteins are generally expected to be similar to random sequences and, accordingly, with no stable tertiary fold and high predicted disorder. However, structural properties of de novo proteins and whether they differ during the stages of emergence and fixation have not been studied in depth and rely heavily on predictions. Here we generated a library of short human putative de novo proteins of varying lengths and ages and sorted the candidates according to their structural compactness and disorder propensity. Using Förster resonance energy transfer combined with Fluorescence-activated cell sorting, we were able to screen the library for most compact protein structures, as well as most elongated and flexible structures. We find that compact de novo proteins are on average slightly shorter and contain lower predicted disorder than less compact ones. The predicted structures for most and least compact de novo proteins correspond to expectations in that they contain more secondary structure content or higher disorder content, respectively. Our experiments indicate that older de novo proteins have higher compactness and structural propensity compared with young ones. We discuss possible evolutionary scenarios and their implications underlying the age-dependencies of compactness and structural content of putative de novo proteins.

Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA.

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 ß-sheets, closely resembling the typical structural topology of most BRCT domains.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Probing Backbone Coupling within Hydrated Proteins with Two-Color 2D Infrared Spectroscopy.

The vibrational coupling between protein backbone modes and the role of water interactions are important topics in biomolecular spectroscopy. Our work reports the first study of the coupling between amide I and amide A modes within peptides and proteins with secondary structure and water contacts. We use two-color two-dimensional infrared (2D IR) spectroscopy and observe cross peaks between amide I and amide A modes. In experiments with peptides with different secondary structures and side chains, we observe that the spectra are sensitive to secondary structure. Water interactions affect the cross peaks, which may be useful as probes for the accessibility of protein sites to hydration water. Moving to two-color 2D IR spectra of proteins, the data demonstrate that the cross peaks integrate the sensitivities of both amide I and amide A spectra and that a two-color detection scheme may be a promising tool for probing secondary structures in proteins.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

Formation of Calprotectin Inhibits Amyloid Aggregation of S100A8 and S100A9 Proteins.

Calcium-binding S100A8 and S100A9 proteins play a significant role in various disorders due to their pro-inflammatory functions. Substantially, they are also relevant in neurodegenerative disorders via the delivery of signals for the immune response. However, at the same time, they can aggregate and accelerate the progression of diseases. Natively, S100A8 and S100A9 exist as homo- and heterodimers, but upon aggregation, they form amyloid-like oligomers, fibrils, or amorphous aggregates. In this study, we aimed to elucidate the aggregation propensities of S100A8, S100A9, and their heterodimer calprotectin by investigating aggregation kinetics, secondary structures, and morphologies of the aggregates. For the first time, we followed the in vitro aggregation of S100A8, which formed spherical aggregates, unlike the fibrillar structures of S100A9 under the same conditions. The aggregates were sensitive to amyloid-specific ThT and ThS dyes and had a secondary structure composed of ß-sheets. Similarly to S100A9, S100A8 protein was stabilized by calcium ions, resulting in aggregation inhibition. Finally, the formation of S100A8 and S100A9 heterodimers stabilized the proteins in the absence of calcium ions and prevented their aggregation.

HYPK: A marginally disordered protein sensitive to charge decoration.

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

Performance of SOBA-AD blood test in discriminating Alzheimer's disease patients from cognitively unimpaired controls in two independent cohorts.

Amyloid-beta (Aß) toxic oligomers are critical early players in the molecular pathology of Alzheimer's disease (AD). We have developed a Soluble Oligomer Binding Assay (SOBA-AD) for detection of these Aß oligomers that contain α-sheet secondary structure that discriminates plasma samples from patients on the AD continuum from non-AD controls. We tested 265 plasma samples from two independent cohorts to investigate the performance of SOBA-AD. Testing was performed at two different sites, with different personnel, reagents, and instrumentation. Across two cohorts, SOBA-AD discriminated AD patients from cognitively unimpaired (CU) subjects with 100% sensitivity, > 95% specificity, and > 98% area under the curve (AUC) (95% CI 0.95-1.00). A SOBA-AD positive readout, reflecting α-sheet toxic oligomer burden, was found in AD patients, and not in controls, providing separation of the two populations, aside from 5 SOBA-AD positive controls. Based on an earlier SOBA-AD study, the Aß oligomers detected in these CU subjects may represent preclinical cases of AD. The results presented here support the value of SOBA-AD as a promising blood-based tool for the detection and confirmation of AD.

Architectonic principles of polyproline II helix bundle protein domains.

Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of α-helices and ß-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers in disordered state ensembles could facilitate glycine-rich polyproline II helix folding, the architectonic bases of these structures are not well known. Here, we compare and analyze their structures to uncover common features. These protein domains are found to be highly tolerant of distinct flanking sequences. This speaks to the robustness of this fold and strongly suggests that glycine rich polyproline II assemblies could be grafted with other protein domains to engineer new structures and functions. These domains are also well packed with few or no cavities. Moreover, a significant trend towards antiparallel helix configuration is observed in all these domains and could provide stabilizing interactions among macrodipoles. Finally, extensive networks of Cα-H···OC hydrogen bonds are detected in these domains. Despite their diverse evolutionary origins and activities, glycine-rich polyproline II helix assemblies share architectonic features which could help design novel proteins.