Secondary Structure Elements Research Articles

Influence of Cataract Causing Mutations on αA-Crystallin: A Computational Approach.

The αA-crystallin protein plays a vital role in maintaining the refractive index and transparency of the eye lens. Significant clinical studies have emerged as the αA-crystallin is prone to aggregation, resulting in the formation of cataracts with varied etiologies due to mutations. This work aims to comprehend the structural and functional role of cataract-causing mutations in αA-crystallin, particularly at N-Terminal and α-Crystallin Domains, using in-silico approaches including molecular dynamics simulation. About 19 mutants of αA-crystallin along with native structure were simulated for 100 ns and the post-simulations analyses reveal pronounced dynamics of αA-crystallin due to the enhanced structure flexibility as its native compactness was lost and is witnessed mainly by the mutants R12L, R21L, R21Q, R54L, R65Q, R116C and R116H. It is observed that αA-crystallin discloses the NTD motions as the dominant one and the same was endorsed by the linear variation between Rg and the center-of-mass of αA-crystallin. Interestingly, such enhanced dynamics of αA-crystallin mutants associated with the structure flexibility is internally modulated by the dynamic exchange of secondary structure elements β-sheets and coils (R2 = 0.619) during simulation. Besides, the observed pronounced dynamics of dimer interface region (β3-L6-β4 segment) of ACD along with CTD dynamics also gains importance. Particularly, the highly dynamic mutants are also characterized by enhanced non-covalent and hydrophobic interactions which renders detrimental effects towards its stability, and favours possible protein unfolding mechanisms. Overall, this study highlights the mutation-mediated structural distortions in αA-crystallin and demands the need for further potential development of inhibitors against cataract formation.

Modifications in Protein Structure and Xanthine Oxidase Inhibition of Yak Casein Induced by Protease Treatment

ABSTRACTYak milk is abundant in casein, which can generate a variety of bioactive peptides through enzymatic hydrolysis. However, the influence of enzymatic hydrolysis on the structural properties of yak casein and its inhibitory effects on xanthine oxidase (XOD) remain largely unexplored. This study demonstrated that when yak casein was subjected to treatment with flavourzyme, the degree of hydrolysis progressively increased over time, resulting in the fragmentation of the casein's flake‐like structure into smaller particles. Circular dichroism analysis revealed that after 4 h of enzymatic treatment, there was an elevation in the β‐sheet content of the yak casein hydrolysate, while other secondary structure elements diminished. Furthermore, flavourzyme treatment induced modifications in the tertiary structure of yak casein. The study also examined the impact of varying hydrolysis durations on XOD inhibitory activity, discovering that the hydrolysate obtained after 3 h displayed the highest inhibition on XOD, with an inhibition rate of (40.63 ± 3.36) %. Additionally, the fraction of the hydrolysate with a molecular weight exceeding 3 kDa demonstrated enhanced XOD inhibitory activity. This study is the first to investigate how varying hydrolysis durations with flavourzyme affect the structural characteristics of yak casein and its XOD inhibitory activity.

Folds from fold: Exploring topological isoforms of a single-domain protein

Expanding the protein fold space beyond linear chains is of fundamental significance, yet remains largely unexplored. Herein, we report the creation of seven topological isoforms (i.e., linear, cyclic, knot, lasso, pseudorotaxane, and catenane) from a single protein fold precursor by rewiring the connectivity of secondary structure elements of the SpyTag-SpyCatcher complex and mutating the reactive residue on SpyTag to abolish the isopeptide bonding. These topological isoforms can be directly expressed in cells. Their topologies were confirmed by combined techniques of proteolytic digestion, fluorescence correlation spectroscopy (FCS), size-exclusion chromatography (SEC), and topological transformation. To study the effects of topology on their structures and properties, their biophysical properties were characterized by differential scanning calorimetry (DSC), heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC-NMR), and circular dichroism (CD) spectroscopy. Molecular dynamics (MD) simulations were further performed to reveal the atomic details of structural changes upon unfolding. Both experimental and simulation results suggest that they share a similar, well-folded hydrophobic core but exhibit distinct folding/unfolding dynamic behaviors. These results shed light onto the folding landscape of topological isoforms derived from the same protein fold. As a model system, this work improves our understanding of protein structure and dynamics beyond linear chains and suggests that protein folds are highly amenable to topological variation.

Visualizing nucleation, condensation and propagation of β-tubulin folding in chaperonin TRiC.

The folding nucleus (FN) initiates protein folding and enables an efficient folding pathway. Here we directly visualize the tubulin FN consisting of a nonnative, partially assembled Rossmann fold, in the closed chamber of human chaperonin TRiC. Chaperonin TRiC interacts with non-natively folded secondary structural elements, stabilizing the nucleus for transition into its first native domain. Through progressive folding, the unfolded sequence goes through drastic spatial arrangement in the TRiC chamber to sample the conformational space, mediated by the highly dynamic CCT tails. The observed presence of individual nonnative secondary structures first in the nonnative FN and then around the incrementally folded native domains supports the hypothesis that tubulin folding in TRiC is a hierarchical process of nucleation, condensation and propagation in cooperation with TRiC subunits.

Raman Microspectroscopy of Hair: Low-Frequency Markers of Protein Secondary Structure.

Low-frequency intervals that can be used to study the secondary structure of proteins are determined. Compared are Ramanspectra of keratins from unpigmented human hair, measured in two experimental configurations: with excitation radiation coaxial with the hair and perpendicular to it. Based on the polarization sensitivity, the bands peaked at 150 and 221 cm-1 are assigned to vibrations of α-helical structures. The comparison of Raman spectra of hair fragments with different contents of secondary structure elements shows that the vibrations of β-structure are manifested in a spectral interval of 270-340 cm-1. The results obtained for a particular object (hair keratin) can be used in the study of the secondary structure of proteins.

Slow Conformational Exchange between Partially Folded and Near-Native States of Ubiquitin: Evidence for a Multistate Folding Model.

The mechanism by which small proteins fold, i.e., via intermediates or via a two-state mechanism, is a subject of intense investigation. Intermediate states in the folding pathways of these proteins are sparsely populated due to transient lifetimes under normal conditions rendering them transparent to a majority of the biophysical methods employed for structural, thermodynamic, and kinetic characterization, which attributes are essential for understanding the cooperative folding/unfolding of such proteins. Dynamic NMR spectroscopy has enabled the characterization of folding intermediates of ubiquitin that exist in equilibrium under conditions of low pH and denaturants. At low pH, an unlocked state defined as N' is in fast exchange with an invisible state, U″, as observed by CEST NMR. Addition of urea to ubiquitin at pH 2 creates two new states F' and U', which are in slow exchange (kF'→U' = 0.14 and kU'→F' = 0.28 s-1) as indicated by longitudinal ZZ-magnetization exchange spectroscopy. High-resolution solution NMR structures of F' show it to be in an "unlocked" conformation with measurable changes in rotational diffusion, translational diffusion, and rotational correlational times. U' is characterized by the presence of just the highly conserved N-terminal β1-β2 hairpin. The folding of ubiquitin is cooperative and is nucleated by the formation of an N-terminal β-hairpin followed by significant hydrophobic collapse of the protein core resulting in the formation of bulk of the secondary structural elements stabilized by extensive tertiary contacts. U' and F' may thus be described as early and late folding intermediates in the ubiquitin folding pathway.

Cyanide mediated conformational changes resulted in the displacement of sulfate ion from the active site of bovine pancreatic ribonuclease A

Ribonuclease A is a major hydrolyzing enzyme involved in the hydrolysis of RNA. The crystals of bovine pancreatic RNase A (bpRNase A) were grown at pH 5.5. The effect of sodium cyanide on bpRNase A was assessed by adding it directly to the crystal containing well. Treating the crystals of bpRNase A with sodium cyanide resulted in the displacement of the sulfate ion from the active site of bpRNase A, while the additional sulfate ion, bound to Ala-4, remained unaffected. The addition of sodium cyanide to bpRNase A crystals did not show change in the secondary structure elements of the enzyme. This study was conducted to check the effect of cyanide on bpRNase A crystals and to displace sulfate ion from its active site.

Enzyme structure correlates with variant effect predictability

Protein engineering increasingly relies on machine learning models to computationally pre-screen promising novel candidates. Although machine learning approaches have proven effective, their performance on prospective screening data leaves room for improvement; prediction accuracy can vary greatly from one protein variant to the next. So far, it is unclear what characterizes variants that are associated with large prediction error. In order to establish whether structural characteristics influence predictability, we created a novel high-order combinatorial dataset for an enzyme spanning 3,706 variants, that can be partitioned into subsets of variants with mutations at positions exclusively belonging to a particular structural class. By training four different supervised variant effect prediction (VEP) models on structurally partitioned subsets of our data, we found that predictability strongly depended on all four structural characteristics we tested; buriedness, number of contact residues, proximity to the active site and presence of secondary structure elements. These dependencies were also found in several single mutation enzyme variant datasets, albeit with dataset specific directions. Most importantly, we found that these dependencies were similar for all four models we tested, indicating that there are specific structure and function determinants that are insufficiently accounted for by current machine learning algorithms. Overall, our findings suggest that improvements can be made to VEP models by exploring new inductive biases and by leveraging different data modalities of protein variants, and that stratified dataset design can highlight areas of improvement for machine learning guided protein engineering.

Rapid Quantification of Protein Secondary Structure Composition from a Single Unassigned 1D 13C Nuclear Magnetic Resonance Spectrum.

The function of a protein is predicated upon its three-dimensional fold. Representing its complex structure as a series of repeating secondary structural elements is one of the most useful ways by which we study, characterize, and visualize a protein. Consequently, experimental methods that quantify the secondary structure content allow us to connect a protein's structure to its function. Here, we introduce an automated gradient descent-based method we refer to as secondary-structure distribution by NMR that allows for rapid quantification of the protein secondary structure composition of a protein from a single, 1D 13C NMR spectrum without chemical shift assignments. The analysis of nearly 900 proteins with known structure and chemical shifts demonstrates the capabilities of our approach. We show that these results rival alternative techniques such as FT-IR and circular dichroism that are commonly used to estimate secondary structure compositions. The resulting method requires only the primary sequence of the protein and its referenced 13C NMR spectrum. Each residue is modeled in an ensemble of secondary structures with percentage contributions from random coil, α-helix, and β-sheet secondary structures obtained by minimizing the difference between a simulated and experimental 1D 13C NMR spectrum. The capabilities of the method are demonstrated as applied to samples at natural abundance or enriched in 13C, acquired by either solution or solid-state NMR, and even on low magnetic field benchtop NMR spectrometers. This approach allows for rapid characterization of protein secondary structure across traditionally challenging to characterize states including liquid-liquid phase-separated, membrane-bound, or aggregated states.

Structural Plasticity within 3-Hydroxy-3-Methylglutaryl Synthases Catalyzing the First Step of β-Branching in Polyketide Biosynthesis Underpins a Dynamic Mechanism of Substrate Accommodation.

Understanding how enzymes have been repurposed by evolution to carry out new functions is a key goal of mechanistic enzymology. In this study we aimed to identify the adaptations required to allow the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) enzymes of primary isoprenoid assembly to function in specialized polyketide biosynthetic pathways, where they initiate β-branching. This role notably necessitates that the HMG synthases (HMGSs) act on substrates tethered to noncatalytic acyl carrier protein (ACP) domains instead of coenzyme A, and accommodation of substantially larger chains within the active sites. Here, we show using a combination of X-ray crystallography and small-angle X-ray scattering, that a model HMGS from the virginiamycin system exhibits markedly increased flexibility relative to its characterized HMGCS counterparts. This mobility encompasses multiple secondary structural elements that define the dimensions and chemical nature of the active site, as well the catalytic residues themselves. This result was unexpected given the well-ordered character of the HMGS within the context of an HMGS/ACP complex, but analysis by synchrotron radiation circular dichroism demonstrates that this interaction leads to increased HMGS folding. This flexible to more rigid transition is notably not accounted for by AlphaFold2, which yielded a structural model incompatible with binding of the native substrates. Taken together, these results illustrate the continued necessity of an integrative structural biology approach combining crystallographic and solution-phase data for elucidating the mechanisms underlying enzyme remodeling, information which can inform strategies to replicate such evolution effectively in the laboratory.

Backbone assignment of CcdB_G100T toxin from E.coli in complex with the toxin binding C-terminal domain of its cognate antitoxin CcdA.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cβ secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

Comparative study of interaction and complex formation of Tremella fuciformis polysaccharide with whey protein, lentil protein, and soy protein isolates

The electrostatic interactions and complex formation of Tremella fuciformis polysaccharide (TPS) with whey protein (WPI), lentil protein (LPI), and soy protein (SPI) isolates were studied. The molecular size and components in each protein isolate complexed with TPS were characterized, along with the structural changes in the protein components, induced by TPS complexation. The stability of emulsions prepared with protein:TPS complexes was also evaluated (pH 4.5). Compared to WPI, the LPI and SPI protein components exhibited more negative charges and larger molecular sizes. Independent of the isolate type, pHc of all protein:TPS systems remained unchanged and pHɸ1 shifted to higher pHs as the protein/TPS ratio increased; the pHopt of WPI:TPS and SPI:TPS occurred at higher values as the protein/TPS ratio increased, while the pHopt of LPI:TPS system was less dependent on the biopolymer mixing ratio, probably due to the lower solubility of LPI. Protein components such as β-LG in WPI, convicilin, vicilin, and basic legumin in LPI, and α’ subunits of 7S β-conglycinin and acidic subunits of 11S glycinin in SPI might be the main components interacting with TPS for complex formation. Different proteins exhibited distinct trends in changing their secondary structural elements when interacting with TPS. At pH 4.5, a high proportion of TPS increased the stability of protein emulsion, regardless of the protein used. The WPI:TPS emulsions with low content of TPS were highly destabilized due to the formation of insoluble WPI:TPS complex coacervates, a phenomenon not observed in emulsions stabilized by the LPI:TPS or SPI:TPS systems.

Identification of novel candidate predisposing genes in familial nonmedullary thyroid carcinoma implicating DNA damage repair pathways.

The genetic basis of nonsyndromic familial nonmedullary thyroid carcinoma (FNMTC) is still poorly understood, as the susceptibility genes identified so far only account for a small percentage of the genetic burden. Recently, germline mutations in DNA repair-related genes have been reported in cases with thyroid cancer. In order to clarify the genetic basis of FNMTC, 94 genes involved in hereditary cancer predisposition, including DNA repair genes, were analyzed in 48 probands from FNMTC families, through targeted next-generation sequencing (NGS). Genetic variants were selected upon bioinformatics analysis and in silico studies. Structural modeling and network analysis were also performed. In silico results of NGS data unveiled likely pathogenic germline variants in 15 families with FNMTC, in genes encoding proteins involved in DNA repair (ATM, CHEK2, ERCC2, BRCA2, ERCC4, FANCA, FANCD2, FANCF, and PALB2) and in the DICER1, FLCN, PTCH1, BUB1B, and RHBDF2 genes. Structural modeling predicted that most missense variants resulted in the disruption of networks of interactions between residues, with implications for local secondary and tertiary structure elements. Functional annotation and network analyses showed that the involved DNA repair proteins functionally interact with each other, within the same DNA repair pathway and across different pathways. MAPK activation was a common event in tumor progression. This study supports that rare germline variants in DNA repair genes may be accountable for FNMTC susceptibility, with potential future utility in patients' clinical management, and reinforces the relevance of DICER1 in disease etiology.

Stapled Peptides: An Innovative and Ultimate Future Drug Offering a Highly Powerful and Potent Therapeutic Alternative.

Peptide-based therapeutics have traditionally faced challenges, including instability in the bloodstream and limited cell membrane permeability. However, recent advancements in α-helix stapled peptide modification techniques have rekindled interest in their efficacy. Notably, these developments ensure a highly effective method for improving peptide stability and enhancing cell membrane penetration. Particularly in the realm of antimicrobial peptides (AMPs), the application of stapled peptide techniques has significantly increased peptide stability and has been successfully applied to many peptides. Furthermore, constraining the secondary structure of peptides has also been proven to enhance their biological activity. In this review, the entire process through which hydrocarbon-stapled antimicrobial peptides attain improved drug-like properties is examined. First, the essential secondary structural elements required for their activity as drugs are validated, specific residues are identified using alanine scanning, and stapling techniques are strategically incorporated at precise locations. Additionally, the mechanisms by which these structure-based stapled peptides function as AMPs are explored, providing a comprehensive and engaging discussion.

Contact-Map-Driven Exploration of Heterogeneous Protein-Folding Paths.

We have recently shown how physically realizable protein-folding pathways can be generated using directed walks in the space of inter-residue contact-maps; combined with a back-transformation to move from protein contact-maps to Cartesian coordinates, we have demonstrated how this approach can generate protein-folding trajectory ensembles without recourse to molecular dynamics. In this article, we demonstrate that this framework can be used to study a challenging protein-folding problem that is known to exhibit two different folding paths which were previously identified through molecular dynamics simulation at several different temperatures. From the viewpoint of protein-folding mechanism prediction, this particular problem is extremely challenging to address, specifically involving folding to an identical nontrivial compact native structure along distinct pathways defined by heterogeneous secondary structural elements. Here, we show how our previously reported contact-map-based protein-folding strategy can be significantly enhanced to enable accurate and robust prediction of heterogeneous folding paths by (i) introducing a novel topologically informed metric for comparing two protein contact maps, (ii) reformulating our graph-represented folding path generation, and (iii) introducing a new and more reliable structural back-mapping algorithm. These changes improve the reliability of generating structurally sound folding intermediates and dramatically decrease the number of physically irrelevant folding intermediates generated by our previous simulation strategy. Most importantly, we demonstrate how our enhanced folding algorithm can successfully identify the alternative folding mechanisms of a multifolding-pathway protein, in line with direct molecular dynamics simulations.

Bridging the Gap between Sequence and Structure Classifications of Proteins with AlphaFold Models

Classification of protein domains based on homology and structural similarity serves as a fundamental tool to gain biological insights into protein function. Recent advancements in protein structure prediction, exemplified by AlphaFold, have revolutionized the availability of protein structural data. We focus on classifying about 9000 Pfam families into ECOD (Evolutionary Classification of Domains) by using predicted AlphaFold models and the DPAM (Domain Parser for AlphaFold Models) tool. Our results offer insights into their homologous relationships and domain boundaries. More than half of these Pfam families contain DPAM domains that can be confidently assigned to the ECOD hierarchy. Most assigned domains belong to highly populated folds such as Immunoglobulin-like (IgL), Armadillo (ARM), helix-turn-helix (HTH), and Src homology 3 (SH3). A large fraction of DPAM domains, however, cannot be confidently assigned to ECOD homologous groups. These unassigned domains exhibit statistically different characteristics, including shorter average length, fewer secondary structure elements, and more abundant transmembrane segments. They could potentially define novel families remotely related to domains with known structures or novel superfamilies and folds. Manual scrutiny of a subset of these domains revealed an abundance of internal duplications and recurring structural motifs. Exploring sequence and structural features such as disulfide bond patterns, metal-binding sites, and enzyme active sites helped uncover novel structural folds as well as remote evolutionary relationships. By bridging the gap between sequence-based Pfam and structure-based ECOD domain classifications, our study contributes to a more comprehensive understanding of the protein universe by providing structural and functional insights into previously uncharacterized proteins.

Natural Lime–Cork Mortar for the Seismic and Energetic Retrofit of Infill Walls: Design, Materials, and Method

Recent seismic events have prompted research into innovative and sustainable materials for strengthening and repairing obsolete and vulnerable buildings. These earthquakes have exposed the high seismic vulnerability of existing reinforced concrete (RC) buildings, particularly in secondary structural elements like infill walls. In addition to structural issues, these buildings often face significant energy deficiencies, such as thermal bridges, due to inadequate insulation. Traditionally, structural and energy improvements for residential buildings are addressed separately with different methods and protocols. This preliminary study is part of a broader research initiative at the University of Reggio Calabria (Italy), aiming to design an innovative fiber-reinforced plaster using natural, sustainable, and locally produced materials to enhance the energy and structural performance of existing wall infills. The study investigates two plaster matrices made of natural hydraulic lime and silica sand, with 15% and 30% cork granules added. Mechanical and thermophysical tests on multiple specimens were conducted to evaluate their suitability for seismic and energy retrofitting of infill walls. Results indicate that adding cork reduces mechanical strength by approximately 42% at a 30% cork content without compromising its use in seismic retrofitting. Thermophysical tests show improved thermal performance with a higher cork content. These findings suggest that the lime–cork mixture at 30% is effective, offering excellent ductility and serving as a promising alternative to traditional cementitious plaster systems. The next experimental phase will test matrices with varying percentages of gorse fiber.

Comprehensive Analysis of the Distinct Nano Environments Characteristics Containing the Different Secondary Structure Elements: Α-Helices, Β-Sheets, and Turns

Comprehensive Analysis of the Distinct Nano Environments Characteristics Containing the Different Secondary Structure Elements: Α-Helices, Β-Sheets, and Turns

Design of a self-regulating mRNA gene circuit

Protein expression in vivo is predominately controlled via regulatory feedback mechanisms that adjust the level of mRNA transcription. However for positive sense single-stranded RNA viruses, protein expression is often controlled via secondary structural elements, such as internal ribosomal entry sites, that are encoded within the mRNA. The self-regulation of mRNA translation observed in this class of viruses suggests that it may be possible to design mRNAs that self-regulate their protein expression, enabling the creation of mRNAs for vaccines and other synthetic biology applications where protein levels in the cell can be tightly controlled without feedback to a transcriptional mechanism. As a proof of concept, I design a polycistronic mRNA based on bacteriophage MS2, where the upstream gene is capable of repressing synthesis of the downstream gene. Using a computational tool that simulates ribosome kinetics and the co-translational folding of the mRNA in response, I show that mutations to the mRNA can be identified which enhance the efficiency of the translation and the repression of the downstream gene. The results of this study open up the possibility of designing bespoke mRNA gene circuits in which the amount of protein synthesised in cells are self-regulated for therapeutic or antigenic purposes.

Universal cold RNA phase transitions

RNA's diversity of structures and functions impacts all life forms since primordia. We use calorimetric force spectroscopy to investigate RNA folding landscapes in previously unexplored low-temperature conditions. We find that Watson-Crick RNA hairpins, the most basic secondary structure elements, undergo a glass-like transition below [Formula: see text]C where the heat capacity abruptly changes and the RNA folds into a diversity of misfolded structures. We hypothesize that an altered RNA biochemistry, determined by sequence-independent ribose-water interactions, outweighs sequence-dependent base pairing. The ubiquitous ribose-water interactions lead to universal RNA phase transitions below TG, such as maximum stability at [Formula: see text]C where water density is maximum, and cold denaturation at [Formula: see text]C. RNA cold biochemistry may have a profound impact on RNA function and evolution.