Alpha-helix Research Articles

Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

Background: The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. Methods: In this study, we used cultured cell models to investigate the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Results: Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. Conclusion: These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

Genetic determinants of SARS-CoV-2 and the clinical outcome of COVID-19 in Southern Bangladesh.

The coronavirus disease 2019 (COVID-19) pandemic has had a severe impact on population health. The genetic determinants ofsevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in southern Bangladesh are not well understood. This study aimed to determine the genomic variation in SARS-CoV-2 genomes that have evolved over 2 years of the pandemic in southern Bangladesh and their association with disease outcomes and virulence of this virus. We investigated demographic variables, disease outcomes of COVID-19 patients and genomic features of SARS-CoV-2. We observed that the disease severity was significantly higher in adults (85.3%) than in children (14.7%), because the expression of angiotensin-converting enzyme-2 (ACE-2) diminishes with ageing that causes differences in innate and adaptive immunity. The clade GK (n = 66) was remarkable between June 2021 and January 2022. Because of the mutation burden, another clade, GRA started a newly separated clustering in December 2021. The burden was significantly higher in GRA (1.5-fold) highlighted in mild symptoms of COVID-19 patients than in other clades (GH, GK, and GR). Mutations were accumulated mainly in S (22.15 mutations per segment) and ORF1ab segments. Missense (67.5%) and synonymous (18.31%) mutations were highly noticed in adult patients with mild cases rather than severe cases, especially in ORF1ab segments. Moreover, we observed many unique mutations in S protein in mild cases compared to severe, and homology modeling revealed that those might cause more folding in the protein's alpha helix and beta sheets. Our study identifies some risk factors such as age comorbidities (diabetes, hypertension, and renal disease) that are associated with severe COVID-19, providing valuable insight regarding prioritizing vaccination for high-risk individuals and allocating health care and resources. The findings of this work outlined the knowledge and mutational basis of SARS-CoV-2 for the next treatment steps. Further studies are needed to confirm the effects of structural and functional proteins of SARS-CoV-2 in detail for monitoring the emergence of new variants in future.

Structural Shifts of the Parvovirus B19 Capsid Receptor-binding Domain: A Peptide Study.

Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by the way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Structural and Functional Analysis of Groundnut bud necrosis virus (GBNV) Using Computational and Biochemical Approaches.

Groundnut bud necrosis virus (GBNV) belonging to the genus Orthotospovirus is transmitted by its vector Thrips palmi. It is a tri-segmented RNA virus that consists of L, M, and S RNA segments. We analysed the secondary structure features of GBNV proteins through various software and predicted the transmembrane helix, glycosylation, and signal peptidase sites within the GBNV protein sequences (GN, GC, N, NSm, and NSs). In glycoprotein sequence, extended strands are predominant (52.87%) whereas the N protein sequence mostly contains alpha helices (47.46%). The random coils are present in movement protein (43.97%) and structural protein (39.41%). We generated the 3D structure of GN and N protein using SWISS MODEL software and quality is validated through PROCHECK and PDBsum software. We also expressed the GBNV proteins (GN, GC, N, NSm, and NSs) in bacterial expression system. The recombinant proteins were used to raise polyclonal antibodies in mice. Our study will be useful in understanding GBNV protein structures in further detail by analysing the important domains that interact with the thrips proteins. This will further aid us in understanding virus-vector relationship through the application of protein-protein interaction and other immunodiagnostic techniques.

A high affinity switch for cAMP in the HCN pacemaker channels

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

In-vitro antimicrobial activity of AF-DP protein and in-silico approach of cell membrane disruption

Microbial resistance against common antibiotics has become one of the most serious threats to human health. The increasing statistics on this problem show the necessity of finding a way to deal with it. In recent years, antimicrobial peptides with unique properties and the capability of targeting a wide range of pathogens, have been considered as a potential for replacing common antibiotics. A small chitin-binding protein with anticandidal activity was isolated from Moringa oleifera seeds by Neto and colleagues in 2017, which very much resembled antimicrobial peptides. In this study, the antimicrobial protein ‘AF-DP’ was identified and characterized. AF-DP was heterologously expressed, purified, and characterized, and its 3D structure was predicted. Six molecular dynamic simulations were performed to investigate how the protein interacts with Gram-negative inner and outer, Gram-positive, fungal, cancerous, and normal mammalian membranes. Also, its antimicrobial and anticancer activity was assessed in vitro via minimum inhibition concentration (MIC) and MTT assays, respectively. This protein with 111 amino acids and a total net charge (of 10.5) has been predicted to be mainly composed of alpha helix and random coils. Its MIC affecting the growth of Escherichia coli, Staphylococcus aureus, and Candida albicans was 30 µg/ml, 100 µg/ml, and 100 µg/ml, respectively; AF-DP showed anticancer activity against MCF-7 breast cancer cell line. Scanning electron microscopic analysis confirmed the creation of pores and scratches on the surface of the bacterial membrane. The results of this research show that AF-DP can be a candidate for the production of new drugs as an AMP with antimicrobial activity. Communicated by Ramaswamy H. Sarma

Inhibition of α-amylase digestion by a Lonicera caerulea berry polyphenol starch complex revealed via multi-spectroscopic and molecular dynamics analyses

Polyphenol-starch complexes exhibit synergistic and beneficial effects on both polyphenols and resistant starches. This study evaluates the inhibitory effects and mechanisms of α-amylase on a Lonicera caerulea berry polyphenol-wheat starch (LPWS) complex following high hydrostatic pressure treatments of 400 MPa for 30 min and 600 MPa for 30 min. The IC50 values for α-amylase inhibition by the complex were 3.61 ± 0.10 mg/mL and 3.42 ± 0.08 mg/mL at a 10 % (w/w) polyphenol content. This interaction was further supported by Fourier-transform infrared spectroscopy and circular dichroism, which confirmed that the alpha helix component of the secondary structure of α-amylase was reduced due to the complex. Multifluorescence spectroscopy revealed that the complex induces changes in the microenvironment of fluorophores surrounding the α-amylase active site. Molecular dynamics simulations and molecular docking revealed that the active site of amylose within the complex becomes enveloped in polyphenol clusters. This wrapping effect reduced the hydrogen bonds between amylose and α-amylase, decreasing from 16 groups to just one group. In summary, the LPWS complex represents a low-digestible carbohydrate food source, thus laying the groundwork for the research and development of functional foods aimed at postprandial hypoglycemic effects.

Impact of steam pressure toasting time on the alpha helix to beta sheet ratios, nutritional value, protein and carbohydrate subfractions, and rumen fermentation parameters of Faba bean seeds for dairy cattle

The use of Faba bean (Vicia faba) seeds (FBS) as a potential feed ingredient for ruminants has been previously evaluated. However, the effects on nutrient supply and degradability linked to protein secondary structure ratios after different steam pressure toasting (SPT) times are scarce. This study aimed to determine the role of varying alpha-helix to beta-sheet ratios induced by SPT duration on the nutritional value of Snowbird FBS in dairy cattle. Fifteen samples (1 variety × 3 years × 5 treatments) were analyzed for protein secondary structures and nutrient composition. The dry matter (DM), crude protein (CP), starch, and neutral detergent fibre-assayed with a heat stable amylase and expressed inclusive of residual ash (aNDF) contents in raw seeds were 914, 286, 328, and 179 g/kg DM, respectively. The averaged DM, CP, starch, and aNDF in the SPT-processed seeds were 904, 292, 374, and 405 g/kg DM, respectively. Soluble crude protein was about five times higher in raw seeds (239 g/kg DM) compared to steam-pressure toasted seeds (avg. 51 g/kg DM) (P < 0.01). The total digestible nutrients (TDN1x) and energy values gradually decreased as SPT time increased (P < 0.01). Hence, the TDN1x in the raw seeds was 832 g/kg DM, while the values at 30, 60, 90, and 120 min were lower by 2.78, 3.87, 6.44, and 7.71 units, respectively. The NEL3x reduced from 2.03 in raw seeds to 1.82 Mcal/kg (P < 0.01). The effective degradability (ED) of nutrients decreased with SPT duration from 0 to 120 min. Hence, the EDDM reduced from 692 to 357 g/kg DM (P < 0.01), the EDCP from 224 to 67 g/kg DM (P < 0.01), and the EDST from 237 to 129 g/kg DM (P = 0.01). Steam pressure toasting from 30 and up to 60 min might be the optimal range for processing FBS (Snowbird var). These findings could be supported by a post-ruminal evaluation of the nutrients that escaped ruminal fermentation.

Genome-wide profile analysis of the Hsp20 family in lettuce and identification of its response to drought stress.

Heat shock protein 20 (Hsp20) plays a very important role in response to abiotic stressors such as drought; however, in lettuce (Lactuca sativa L.), this gene family is poorly understood. This study used bioinformatics methods to identify 36 members of the lettuce Hsp20 family, which were named LsHsp20-1~LsHsp20-36. Subcellular localization results revealed that 26 members of the LsHsp20 protein family localized to the cytoplasm and nucleus. Additionally, 15 conserved domains were identified in the LsHsp20 protein family, with the number of amino acids ranging from 8 to 50. Gene structure analysis revealed that 15 genes (41.7%) had no introns, and 20 genes (55.5%) had one intron. The proportion of the LsHsp20 secondary structure was random coil > alpha helix > extended strand > beta turn. Chromosome positioning analysis indicated that 36 genes were unevenly distributed on nine chromosomes, and four pairs of genes were collinear. The Ka/Ks ratio of the collinear genes was less than 1, indicating that purifying selection dominated during L. sativa evolution. Thirteen pairs of genes were collinear in lettuce and Arabidopsis, and 14 pairs of genes were collinear in lettuce and tomato. A total of 36 LsHsp20 proteins were divided into 12 subgroups based on phylogenetic analysis. Three types of cis-acting elements, namely, abiotic and biotic stress-responsive, plant hormone-responsive, and plant development-related elements, were identified in the lettuce LsHsp20 family. qRT-PCR was used to analyze the expression levels of 23 LsHsp20 genes that were significantly upregulated on the 7th or 14th day of drought treatment, and the expression levels of two genes (LsHsp20-12 and LsHsp20-26) were significantly increased by 153-fold and 273-fold on the 14th and 7th days of drought treatment, respectively. The results of this study provide comprehensive information for research on the LsHsp20 gene family in lettuce and lay a solid foundation for further elucidation of Hsp20 biological functions, providing valuable information on the regulatory mechanisms of the LsHsp20 family in lettuce drought resistance.

Quasi-spherical silver nanoparticles for human prolactin detection by surface-enhanced Raman spectroscopy.

Prolactin is a polypeptide hormone made of 199 amino acids; 50% of the amino acid chain forms helices, and the rest forms loops. This hormone is typically related to initiating and maintaining lactation, although it is also elevated in various pathological conditions. Serum prolactin levels of 2 to 18 ng ml-1 in men, up to 30 ng ml-1 in women, and 10 to 210 ng ml-1 in pregnant women are considered normal. Immunoassay techniques used for detection are susceptible to error in different clinical conditions. Surface-enhanced Raman spectroscopy (SERS) is a technique that allows for obtaining the protein spectrum in a simple, fast, and reproducible manner. Nonetheless, proper characterization of human prolactin's Raman/SERS spectrum at different concentrations has so far not been deeply discussed. This study aims to characterize the Raman spectrum of human prolactin at physiological concentrations using silver nanoparticles (AgNPs) as the SERS substrate. The Raman spectrum of prolactin at 20 ng ul-1 was acquired. Quasi-spherical AgNPs were obtained using chemical synthesis. For SERS characterization, decreasing dilutions of the protein were made by adding deionized water and then a 1 : 1 volume of the AgNPs colloid. For each mixture, the Raman spectrum was determined. The spectrum of prolactin by SERS was obtained with a concentration of up to 0.1 ng ml-1. It showed characteristic bands corresponding to the side chains of aromatic amino acids in the protein's primary structure and the alpha helices of the secondary structure of prolactin. In conclusion, using quasi-spherical silver nanoparticles as the SERS substrate, the Raman spectrum of human prolactin at physiological concentration was determined.

In-silico structural analysis of Heterocephalus glaber amyloid beta: an anti-Alzheimer's peptide.

Heterocephalus glaber, known as the Naked mole-rat, has an extraordinary immunity to Alzheimer's disease. The pathological hallmark of Alzheimer's disease is cerebral accumulations of plaques, consisting of self-aggregated amyloid beta peptides. Homo sapiens and H. glaber amyloid beta peptides are different in only one amino acid. Herein, computational structural analyses were carried out to determine whether plaque development in H. glaber is prevented by the replacement of His13 with Arg13 in the amyloid beta peptide. AlphaFold2 was used to predict the structure of the H. glaber amyloid beta peptide. HADDOCK and Hex were used to self-dock the peptides and dock ions on peptides, respectively. Illustrations were made by PyMol and ChimeraX. Using VMD, we calculated the radius of gyration. The phylogenetic analysis was conducted by Mega. The results showed an accurate structure with two alpha helices separated by a short coil for H. glaber. Self-docking of the two amyloid beta peptides demonstrated a globular conformation in the H. glaber dimer, implying the unlikeliness of amyloid beta peptides' self-aggregation to form fibrillar structures. This conformational state resulted in lower electrostatic energy compared to H. sapiens, contributing to H. glaber's lower tendency for fibril and, ultimately, plaque formation. Phylogenetic analysis confirmed that amyloid precursor protein is highly conserved in each taxon of rodentia and primata. This study provides insight into the connection between the structure of H. glaber amyloid beta and its plaque formation properties, showing that the Arg13 in H. glaber leads to fibril instability, and might prevent senile plaque accumulation.

Классификация и характеристика нового антимикробного пептида

In recent years, significant progress has been achieved in AMP research, revealing the potential of these compounds as alternatives to traditional antibiotics. Due to the diverse mechanisms of action and a wide range of antibacterial activity, AMP provides new opportu­nities for preventing and treating multidrug-resistant infections by creating foods with anti­microbial properties, new antimicrobial biologically active additives and medicines. This study aimed to create, characterise and classify a new AMP. The DRAMP proteo­mic database was used to design the AMP, and the Peptide Ranker was utilised to predict biological activity (http://distilldeep.ucd.ie/PeptideRanker). The peptide was identified with the help of the EROP-Moscow database (http://erop.inbi.ras.ru/index.html), and its physio-chemical properties were studied using the APD, DRAMP and PepCalc databases (http://pepcalc.com). The research resulted in the design of a new AMP consisting of 11 ami­no acid residues with the following percentages: Val, Ala, Trp, Gly, Pro, Tyr, Glu, His, Lys by 9 % and Arg by 18 %. The total hydrophobic ratio in the peptide, determined by APD, is 27 %; the total net charge is +2.25. Its molecular formula is C64H96N21O15S0, with protein binding potential (Boman index) at 3.44 kcal/mol. The peptide can form an alpha helix. The resulting peptide can be classified as a synthesised cationic hydrophobic alpha-helical antimi­crobial polypeptide.

Structural bioinformatics studies of six human ABC transporters and their AlphaFold2-predicted water-soluble QTY variants.

Human ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins and perform diverse functions. Many of them are associated with multidrug resistance that often results in cancer treatment with poor outcomes. Here, we present the structural bioinformatics study of six human ABC membrane transporters with experimentally determined cryo-electron microscopy (CryoEM) structures including ABCB7, ABCC8, ABCD1, ABCD4, ABCG1, ABCG5, and their AlphaFold2-predicted water-soluble QTY variants. In the native structures, there are hydrophobic amino acids such as leucine (L), isoleucine (I), valine (V), and phenylalanine (F) in the transmembrane alpha helices. These hydrophobic amino acids are systematically replaced by hydrophilic amino acids glutamine (Q), threonine (T), and tyrosine (Y). Therefore, these QTY variants become water soluble. We also present the superposed structures of native ABC transporters and their water-soluble QTY variants. The superposed structures show remarkable similarity with root mean square deviations between 1.064 and 3.413Å despite significant (41.90-54.33%) changes to the protein sequence of the transmembrane domains. We also show the differences in hydrophobicity patches between the native ABC transporters and their QTY variants. We explain the rationale behind why the QTY membrane protein variants become water soluble. Our structural bioinformatics studies provide insight into the differences between the hydrophobic helices and hydrophilic helices and will likely further stimulate designs of water-soluble multispan transmembrane proteins and other aggregated proteins. The water-soluble ABC transporters may be useful as soluble antigens to generate therapeutic monoclonal antibodies for combating multidrug resistance in clinics.

Jujube Witches' Broom Phytoplasma Effector Zaofeng3, a Homologous Effector of SAP54, Induces Abnormal Floral Organ Development and Shoot Proliferation.

Plant-pathogenic phytoplasmas secrete specific virulence proteins into a host plant to modulate plant function for their own benefit. Identification of phytoplasmal effectors is a key step toward clarifying the pathogenic mechanisms of phytoplasma. In this study, Zaofeng3, also known as secreted jujube witches' broom phytoplasma protein 3 (SJP3), was a homologous effector of SAP54 and induced a variety of abnormal phenotypes, such as phyllody, malformed floral organs, witches' broom, and dwarfism in Arabidopsis thaliana. Zaofeng3 can also induce small leaves, dwarfism, and witches' broom in Ziziphus jujuba. Further experiments showed that the three complete α-helix domains predicted in Zaofeng3 were essential for induction of disease symptoms in jujube. Yeast two-hybrid library screening showed that Zaofeng3 mainly interacts with proteins involved in flower morphogenesis and shoot proliferation. Bimolecular fluorescence complementation assays confirmed that Zaofeng3 interacted with these proteins in the whole cell. Overexpression of zaofeng3 in jujube shoot significantly altered the expression patterns of ZjMADS19, ZjMADS47, ZjMADS48, ZjMADS77, and ZjTCP7, suggesting that overexpressing zaofeng3 might induce floral organ malformation and witches' broom by altering the expression of the transcriptional factors involved in jujube morphogenesis.

Comprehensive Network and Structural Analysis of Bovine Papillomavirus, Squamous Cell Carcinoma Markers, and Elucidation of Efficacy Mechanisms of Phytochemicals from Thuja Occidentalis

Papillomaviruses infect cutaneous tissue in various species including bovines and from benign warts to malignant squamous cell carcinoma causing severe economic losses to the farmers. The mechanisms by which bovine papillomaviruses interact with host tissue are unclear. Hence in this study using classical network analysis tools, we evaluated interactions of Bovine papilloma (BPV) variants, with markers and receptors implicated in squamous cell carcinoma. Additionally, the Thuja phytoconstituents were also evaluated for its potential to target the BPV and squamous cell carcinoma network interactions to understand the mechanism of its clinical benefits. Various protein composition of 14 different virus variants of BPV were assessed against 24 markers of squamous cell carcinoma. Among these interactions EGFR consistently exhibited high-affinity interactions with the E1 protein in all isoforms of BPV. Type 4 BPV displayed the maximum number of binding sites (14) with a binding pocket score ranging from 15.47 to 141.34 and a probability score of 0.75 to 0.99. The comparison of the binding pockets identified that BPV types 2 and 13 had the highest number of common amino acid sequences. Further the alpha helix structure of specific common amino acid sequences contributes to a more robust and widespread affinity interaction with both E1 of various BPV types and EGFR. Analysis of Thuja phytochemicals suggested superior efficacy of Beyerene and Terpinene-4-ol towards all ten BPV targets and bEGFR. In conclusion, our comprehensive study leading to identification of E1 protein of BPV as a major interacting network with bEGFR, their key binding sites, and efficacy of Thuja phytoconstituents offer valuable insight into further experimental validation and development of novel therapeutic strategies against BPV-associated diseases.

Разработка нового антимикробного пептида

The properties of antimicrobial peptides as substances with high biological activity are considered; they can also be used as functional ingredients and (or) preservative substances in food product composition for preventive and specialized purposes. A new antimicrobial peptide consisting of 20 amino acid residues was developed; during the work, suitable amino acid sequences were selected, physical and chemical characteristics were optimized, and activity against target microorganisms was assessed. The inclusion of positively charged amino acids, such as lysine and arginine, which increase the cationicity of antimicrobial peptides and therefore increase their selectivity towards microbial membranes, is considered. The percentage content of each amino acid is as follows, %: Val – 5, Leu – 25, Cys – 5, Ala – 20, Trp – 5, Gly – 5, Ser – 5, His – 5, Lys – 20 and Arg – 5. General hydrophobicity ratio determined by APD is 60%, the charge is + 5.25. The peptide has a molecular weight of 2 206.761 Da, protein binding potential (Bohmann index) of 0.23 kcal/mol. The peptide can form alpha helices and has at least 8 elements on a single hydrophobic surface. According to data obtained from the APD database, the peptide can interact with bacterial membranes and is antimicrobial. The following known antimicrobial peptides are closest in physical and chemical characteristics to the created peptide: AP 00500, AP 03880 and AP 03383. The developed peptide can be classified as a cationic alpha-helical hydrophobic antimicrobial peptide.

IN-SILICO CHARACTERIZATION OF HUMAN HER2 GENE TO PREDICT THE BREAST CANCER ASSOCIATED BIOMARKERS

HER2 gene hyperactivation is responsible for the incidence of invasive breast cancer (BC). It leads to over expression of HER2 receptors in breast cells resulting in abnormal receptor dimerization and signal transduction cascade initiation. It ultimately causes uncontrolled proliferation of breast cells. Therefore, this is the most susceptible target gene for BC treatment. HER2 gene transcript with accession ID ENST00000269571.10 was selected for association analysis in present study. A total of ten single nucleotide polymorphisms (SNPs) of gene were analyzed using in-silico tools like CELLO, PROTPARAM, SOPMA, and SWISSMODEL. These tools helped in determining the effect of variants on sub-cellular localization, physiochemical properties, and secondary (2D) and tertiary (3D) structures of mutated proteins. Variants rs1249755832, rs769703068, rs1567913219, and rs1213602748 altered the localization of mutated proteins. SNPs rs1213602748, rs769703068, and rs1249755832 considerably changed the isoelectric point (pI), extinction coefficient, instability index, aliphatic index, and GRAVY. Variant rs769703068 caused a change in all four properties of the 2D structure, i.e., alpha helix, extended strand, beta-turn, and random coil. Polymorphisms that caused a significant deviation in 3D configuration were rs769703068, rs144434331, and rs146177313. These variants can be recommended as biomarkers for HER2+ BC cell diagnosis.

Structural modelling and functional analysis of Pseudomonas ACC deaminase: An in silico study

The activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme and its impact on regulating ethylene concentrations and plant ACC are fundamental characteristic found in plant-associated rhizobacteria. ACC deaminase-producing bacteria, such as Pseudomonas mitigate the detrimental effects of high levels of ethylene and ACC in plant-microbe interactions, resulting in improved plant growth and development. Different species within the Pseudomonas genus are commonly found in plant microbiome worldwide. Their adaptation to the plant-associated environment makes several Pseudomonas strains highly promising for advancing novel sustainable biotechnological and agricultural solutions, particularly those exhibiting ACC deaminase activity. Thus, the present study conducted a comprehensive in silico analysis focusing on the structural and functional characteristics of ACC deaminase protein of Pseudomonas spp. The study explored correlations based on phylogenetic relatedness using both ACC deaminase enzymes and their respective genes across different Pseudomonas species and strains. This study also investigated physiochemical properties, CATH classification and STRING analysis using various bioinformatics tools. Pseudomonas brassicacearum was selected as the representative species from the Pseudomonas genus for the 3D modeling of ACC deaminase protein. The acidic ACC deaminase protein has an average molecular weight of around 25.30 kDa, with a high percentage of alpha helices in its secondary structure, demonstrating its thermal stability. This theoretical assessment of the structure and function of ACC deaminase-producing Pseudomonas could assist researchers in understanding the ACC deaminase protein, potentially facilitating a better understanding of plant-microbe interactions, and aiding in the selection of highly efficient strains for various agricultural applications.

Comprehensive bioinformatics-based annotation and functional characterization of bovine chymosin protein revealed novel biological insights

Chymosin, an aspartic protease present in the stomachs of young ruminants like cows (bovine), causes milk coagulation and cheese production through the breakdown of κ-casein peptide bonds at the Met105-Phe106 site. Bovine chymosin is first synthesized as a pre-prochymosin that is cleaved to produce the mature chymosin protein. Despite significant strides in research, our understanding of this crucial enzyme remains incomplete. The purpose of this work was to perform in silico evolutionary and functional analysis and to gain unique insights into the structure of this protein. For this, the sequence of Bos taurus chymosin from UniProt database was subjected to various bioinformatics analyses. We found that bovine chymosin is a low molecular weight and hydrophilic protein that has homologs in other Bovidae species. Two active sites of aspartic peptidases, along with a functional domain, were identified. Gene Ontology analysis further confirmed chymosin's involvement in proteolysis and aspartic endopeptidase activity. Potential disordered residues and post-translational modification sites were also uncovered. It was revealed that the secondary structure of bovine chymosin is comprised of beta strands (44.27%), coils (43.65%), and alpha helices (12.07%). A highly optimized 3D structure was also obtained. Moreover, crucial protein–protein interactions were unveiled. Altogether, these findings provide valuable insights that could guide future research on bovine chymosin and its biological roles.

Molecular dynamics simulations at high temperatures of the Aeropyrum pernix L7Ae thermostable protein: Insight into the unfolding pathway

Most life forms on earth live at temperatures below 50 °C. Within these organisms are proteins that form the three-dimensional structures essential to their biological activity and function. However, some thermophilic life forms can resist higher temperatures and have corresponding adaptations to preserve protein function at these high temperatures. Among the structural factors responsible for this resistance of thermophilic proteins to high temperatures is the presence of additional hydrogen bonds in the thermophilic proteins, which means that the structure of the protein is more resistant to unfolding. Similarly, thermostable proteins are rich in structure-stabilizing salt bridges and/or disulfide bridges. In this context, we perform multiple replica molecular dynamics simulations at different temperatures on the Aeropyrum pernix (L7Ae) protein (from the crenarchaeal species A. pernix), known for its high melting temperature, and this in the aim to elucidate the structural factors responsible for its high thermostability. The results reveal that between the most sensitive regions of the protein to the increase of temperature are the loops L1, and L5, which surround the hydrophobic core region of the protein, besides the loop L9, and the C-terminal α5 region. This latter is the longer alpha helix of the protein secondary structure motifs and it is the first to be denaturated at 450 K, while the rest of the protein secondary structure motifs at this temperature were intact. The mechanism of unfolding that follows this protein at 550 K is similar to other thermophile proteins found in literature, with the opening of the loops that surround the hydrophobic core of the protein. So, the latter is completely exposed to the solvent, and partially denatured. The total denaturation process of the protein takes an average time of 40 ns to be achieved. Our investigation also shows that all the calculated salt bridges, with distances less than or equal to 6 A°, are on the periphery part of the protein, exposed to the solvent. However, the hydrophobic core of the protein is not involved in the formation of salt bridges, but rather with formation of some important hydrogen bondings that still persist even at 450 K. So, optimizing hydrogen bonding, near or within the core region, at high temperatures is a strategy that follows this thermostable protein to protect its hydrophobic core from denaturation, and ensure the thermal stability of the protein.