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International Journal of Molecular... Feb 2022Nuclear-encoded Atp23 was previously shown to have dual functions, including processing the yeast Atp6 precursor and assisting the assembly of yeast mitochondrial ATP...
Nuclear-encoded Atp23 was previously shown to have dual functions, including processing the yeast Atp6 precursor and assisting the assembly of yeast mitochondrial ATP synthase. However, it remains unknown whether there are genes functionally complementary to to rescue null mutant. In the present paper, we screen and characterize three revertants of null mutant and reveal a T1121G point mutation in the mitochondrial gene coding sequence, which leads to Val374Gly mutation in Cox1, the suppressor in the revertants. This was verified further by the partial restoration of mitochondrial ATP synthase assembly in null mutant transformed with exogenous hybrid mutant plasmid. The predicted tertiary structure of the Cox1 p.Val374Gly mutation showed no obvious difference from wild-type Cox1. By further chase labeling with isotope [S]-methionine, we found that the stability of Atp6 of ATP synthase increased in the revertants compared with the null mutant. Taking all the data together, we revealed that the T1121G point mutation of mitochondrial gene could partially restore the unassembly of mitochondrial ATP synthase in null mutant by increasing the stability of Atp6. Therefore, this study uncovers a gene that is partially functionally complementary to to rescue deficiency, broadening our understanding of the relationship between yeast the cytochrome c oxidase complex and mitochondrial ATP synthase complex.
Topics: Adenosine Triphosphate; Amino Acid Sequence; DNA, Mitochondrial; Electron Transport Complex IV; Genes, Mitochondrial; Loss of Function Mutation; Metalloproteases; Mitochondria; Mitochondrial Proton-Translocating ATPases; Point Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35216443
DOI: 10.3390/ijms23042327 -
Methods in Molecular Biology (Clifton,... 2020The rational design of enzymes is a challenging research field, which plays an important role in the optimization of a wide series of biotechnological processes....
The rational design of enzymes is a challenging research field, which plays an important role in the optimization of a wide series of biotechnological processes. Computational approaches allow screening all possible amino acid substitutions in a target protein and to identify a subset likely to have the desired properties. They can thus be used to guide and restrict the huge, time-consuming search in sequence space to reach protein optimality. Here we present HoTMuSiC, a tool that predicts the impact of point mutations on the protein melting temperature, which uses the experimental or modeled protein structure as sole input and is available at the dezyme.com website. Its main advantages include accuracy and speed, which makes it a perfect instrument for thermal stability engineering projects aiming at designing new proteins that feature increased heat resistance or remain active and stable in nonphysiological conditions. We set up a HoTMuSiC-based pipeline, which uses additional information to avoid mutations of functionally important residues, identified as being too well conserved among homologous proteins or too close to annotated functional sites. The efficiency of this pipeline is successfully demonstrated on Rhizomucor miehei lipase.
Topics: Amino Acid Substitution; Enzyme Stability; Hot Temperature; Lipase; Point Mutation; Protein Engineering; Protein Stability; Proteins; Rhizomucor; Temperature
PubMed: 32006278
DOI: 10.1007/978-1-0716-0270-6_5 -
Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.Scientific Reports May 2022Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant...
Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.
Topics: CRISPR-Cas Systems; Catalysis; Gene Editing; Mutagenesis; Mutation; Point Mutation
PubMed: 35581233
DOI: 10.1038/s41598-022-11808-2 -
The Journal of Craniofacial SurgeryMicrotia is a congenital malformation of the external ear that can lead to conductive hearing impairment. In this study, we investigated the role of the Prkra gene in...
Microtia is a congenital malformation of the external ear that can lead to conductive hearing impairment. In this study, we investigated the role of the Prkra gene in external ear development. We used advanced sequencing techniques to evaluate the differential expression of microRNAs (miRNAs) involved in external ear development in mouse embryos after point mutation in the Prkra gene. The Prkra Little ear mouse model was used to obtain mouse embryos at the E15.5 and E17.5 developmental stages, and changes in miRNA expression profiles were detected. Gene ontology and Kyoto Encyclopedia of Genes and Genomes functional annotations were performed on differentially expressed miRNAs, and existing and new miRNAs were studied. miRNAs were observed to be involved in multiple signaling pathways during the E15.5 and E17.5 developmental stages. The results show a correlation between miRNA regulation and external ear development in Prkra Little ear mice, and differences were detected in key regulatory miRNAs owing to point mutations in the Prkra gene. This study provides new insights into the biological mechanisms through which miRNAs regulate external ear development in mouse embryos. Changes in the mouse miRNA expression profiles can also provide insights into the pathogenesis of human congenital microtia at the level of miRNA regulation.
Topics: Humans; Mice; Animals; MicroRNAs; Point Mutation; Embryonic Development; Ear, External; Congenital Microtia; Gene Expression Profiling; RNA-Binding Proteins
PubMed: 35968958
DOI: 10.1097/SCS.0000000000008837 -
International Journal of Molecular... Dec 2022Antibiotic resistance has been becoming more and more critical due to bacteria's evolving hydrolysis enzymes. The NDM-1 enzyme could hydrolyze not only carbapenems but...
The Effects of One-Point Mutation on the New Delhi Metallo Beta-Lactamase-1 Resistance toward Carbapenem Antibiotics and β-Lactamase Inhibitors: An In Silico Systematic Approach.
Antibiotic resistance has been becoming more and more critical due to bacteria's evolving hydrolysis enzymes. The NDM-1 enzyme could hydrolyze not only carbapenems but also most of β-lactam's antibiotics and inhibitors. In fact, variant strains could impose a high impact on the resistance of bacteria producing NDM-1. Although previous studies showed the effect of some variants toward antibiotics and inhibitors binding, there has been no research systematically evaluating the effects of alternative one-point mutations on the hydrolysis capacity of NDM-1. This study aims to identify which mutants could increase or decrease the effectiveness of antibiotics and β-lactamase inhibitors toward bacteria. Firstly, 35 different variants with a high probability of emergence based on the PAM-1 matrix were constructed and then docked with 5 ligands, namely d-captopril, l-captopril, thiorphan, imipenem, and meropenem. The selected complexes underwent molecular dynamics simulation and free energy binding estimation, with the results showing that the substitutions at residues 122 and 124 most influenced the binding ability of NDM-1 toward inhibitors and antibiotics. The H122R mutant decreases the binding ability between d-captopril and NDM-1 and diminishes the effectiveness of this antibiotic toward Enterobacteriaceae. However, the H122R mutant has a contrary impact on thiorphan, which should be tested in vitro and in vivo in further experiments.
Topics: Carbapenems; beta-Lactamase Inhibitors; Point Mutation; Captopril; Thiorphan; Anti-Bacterial Agents; beta-Lactamases; Bacteria; Microbial Sensitivity Tests
PubMed: 36555726
DOI: 10.3390/ijms232416083 -
Plant Disease Oct 2019Fusarium head blight, also called scab, is caused by and is one of the most important destructive diseases of wheat. The frequency of carbendazim resistance in 1,132...
Fusarium head blight, also called scab, is caused by and is one of the most important destructive diseases of wheat. The frequency of carbendazim resistance in 1,132 isolates of recovered from fields in different regions of Henan Province in 2016, 2017, and 2018 was determined. A total of 31 isolates resistant to carbendazim were detected, including 30 moderately resistant isolates and one highly resistant isolate. The frequency of resistance of isolates to carbendazim was 2.7%. The range of effective concentration (EC) values of 1,101 sensitive isolates and 30 moderately resistant isolates was 0.08 to 0.98 μg ml and 2.73 to 13.28 μg ml, respectively. The mean ± SD EC value was 0.55 ± 0.13 μg ml and 5.61 ± 2.58 μg ml, respectively. The EC value of the highly resistant isolate was 21.12 μg ml. Point mutation types of the carbendazim-resistant isolates were characterized by cloning the β-tubulin gene of 31 resistant isolates. Three point mutation types at amino acids F167Y, E198Q, and E198L in the β-tubulin gene of resistant isolates were identified. Among 31 resistant isolates, the frequency of point mutation types in F167Y, E198Q, and E198L of the β-tubulin gene was 71.0, 25.8, and 3.2%, respectively. The data indicate that has developed resistance to carbendazim in Henan Province, and single point mutations at amino acid F167Y were the predominant type of mutation detected.
Topics: Benzimidazoles; Carbamates; Drug Resistance, Fungal; Fungicides, Industrial; Fusarium; Genes, Plant; Point Mutation; Triticum; Tubulin
PubMed: 31424998
DOI: 10.1094/PDIS-02-19-0391-RE -
Nucleic Acids Research Jul 2023Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite...
Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.
Topics: Deep Learning; Mutation; Point Mutation; Protein Stability; Software; Proteins
PubMed: 37283042
DOI: 10.1093/nar/gkad472 -
Scientific Reports May 2022Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of...
Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of "mox"-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pK values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pK value is the C-O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C-O bond lengths are found for proteins with larger pK. This feature can be utilized for prediction of the pK values of red fluorescent proteins.
Topics: Coloring Agents; Cysteine; Green Fluorescent Proteins; Luminescent Proteins; Mutagenesis; Oxidation-Reduction; Point Mutation
PubMed: 35551209
DOI: 10.1038/s41598-022-11249-x -
Bio Systems Oct 2021Many theories of the evolution of the genetic code assume that the genetic code has always evolved in the direction of increasing the supply of amino acids to be encoded...
Many theories of the evolution of the genetic code assume that the genetic code has always evolved in the direction of increasing the supply of amino acids to be encoded (Barbieri, 2019; Di Giulio, 2005; Wong, 1975). In order to reduce the risk of the formation of a non-functional protein due to point mutations, nature is said to have built in control mechanisms. Using graph theory the authors have investigated in Blazej et al. (2019) if this robustness is optimal in the sense that a different codon-amino acid assignment would not generate a code that is even more robust. At present, efforts to expand the genetic code are very relevant in biotechnological applications, for example, for the synthesis of new drugs (Anderson et al., 2004; Chin, 2017; Dien et al., 2018; Kimoto et al., 2009; Neumann et al., 2010). In this paper we generalize the approach proposed in Blazej et al. (2019) and will explore hypothetical extensions of the standard genetic code with respect to their optimal robustness in two ways: (1) We keep the usual genetic alphabet but move from codons to longer words, such as tetranucleotides. This increases the supply of coding words and thus makes it possible to encode non-canonical amino acids. (2) We expand the genetic alphabet by introducing non-canonical base pairs. In addition, the approach from Blazej et al. (2019) and Blazej et al. (2018) is extended by incorporating the weights of single point-mutations into the model. The weights can be interpreted as probabilities (appropriately normalized) or degrees of severity of a single point mutation. In particular, this new approach allows us to take a closer look at the wobble effects in the translation of codons into amino acids. According to the results from Blazej et al. (2019) and Blazej et al. (2018), the standard genetic code is not optimal in terms of its robustness to point mutations if the weights of single point mutations are not taken into account. After incorporation into the model weights that mimic the wobble effect, the results of the present work show that it is much more robust, almost optimal in that respect. We hope, that this theoretical analysis might help to assess extended genetic codes and their abilities to encode new amino acids.
Topics: Animals; Codon; Evolution, Molecular; Genetic Code; Humans; Models, Genetic; Point Mutation
PubMed: 34280517
DOI: 10.1016/j.biosystems.2021.104485 -
Analytical Chemistry Aug 2020Point mutations are a common form of genetic variation and have been identified as important disease biomarkers. Conventional methods for analyzing point mutations,...
Point mutations are a common form of genetic variation and have been identified as important disease biomarkers. Conventional methods for analyzing point mutations, e.g., polymerase chain reaction (PCR), are based on differences in thermal stability of the DNA duplex, which require extensive optimization of the reaction condition and nontrivial design of sequence-selective primers. This motivated the design of molecular translators to convert molecular inputs into generic output sequences, which allows for the target recognition and signal generation regions to be designed independently. In this work, we propose a translator design based on the concept of split proximity circuit (SPC) to achieve both high sequence selectivity and assay robustness using a universal reaction condition, i.e., room temperature and constant ionic concentration. We discussed the design aspects of the SPC recognition regions and demonstrated its plug-and-play capability to discriminate different point mutations for both DNA (seven G6PD mutations) and RNA (let-7 microRNA family members) targets while retaining the same signal generation region. Despite its simple design and nonstringent assay condition requirements, the SPC retained good analytical performance to detect subnanomolar target concentration within a reasonable time of an hour.
Topics: DNA; Glucosephosphate Dehydrogenase; Humans; Limit of Detection; MicroRNAs; Point Mutation; Polymerase Chain Reaction
PubMed: 32605366
DOI: 10.1021/acs.analchem.0c01379