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Nature Chemical Biology Jan 2015Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this...
Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.
Topics: Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Point Mutation; Protein Binding; Protein Conformation; Substrate Specificity; Ubiquinone; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 25436519
DOI: 10.1038/nchembio.1700 -
Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries.The Journal of Biological Chemistry Apr 2022Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains...
Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, especially for proteins that are well folded and relatively stable. Here, we demonstrate that incorporation of a single, specific, destabilizing mutation termed parent inactivating mutation into each member of a single-site saturation mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. We carried out fluorescence-activated cell sorting of such a yeast surface display, saturation suppressor library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations. We found that multiple stabilizing mutations could be identified after a single round of sorting. In addition, multiple libraries with different parent inactivating mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Finally, we show that individual stabilizing mutations could be combined to result in a multi-mutant that demonstrated an increase in thermal melting temperature of about 20 °C, and that displayed enhanced tolerance to high temperature exposure. We conclude that as this method is robust and employs small library sizes, it can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.
Topics: Mutagenesis; Point Mutation; Protein Stability; Proteins; Saccharomyces cerevisiae
PubMed: 35247389
DOI: 10.1016/j.jbc.2022.101785 -
Biophysical Journal Oct 2020Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of...
Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of reversible oligomers at high temperature (N↔I↔D). This contrasts sharply with the standard two-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, and Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect two-state denaturation, as evidenced by the single endothermic peak and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. Furthermore, our present study showing that a point mutation, L342A, designed based on the crystal structure inhibited RO is surprising because RO occurs at a high-temperature. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.
Topics: Calorimetry, Differential Scanning; Hot Temperature; Point Mutation; Protein Denaturation; Temperature; Thermodynamics
PubMed: 32961107
DOI: 10.1016/j.bpj.2020.08.023 -
Molecular Biology and Evolution Sep 2017While mutational biases strongly influence neutral molecular evolution, the role of mutational biases in shaping the course of adaptation is less clear. Here we consider...
While mutational biases strongly influence neutral molecular evolution, the role of mutational biases in shaping the course of adaptation is less clear. Here we consider the frequency of transitions relative to transversions among adaptive substitutions. Because mutation rates for transitions are higher than those for transversions, if mutational biases influence the dynamics of adaptation, then transitions should be overrepresented among documented adaptive substitutions. To test this hypothesis, we assembled two sets of data on putatively adaptive amino acid replacements that have occurred in parallel during evolution, either in nature or in the laboratory. We find that the frequency of transitions in these data sets is much higher than would be predicted under a null model where mutation has no effect. Our results are qualitatively similar even if we restrict ourself to changes that have occurred, not merely twice, but three or more times. These results suggest that the course of adaptation is biased by mutation.
Topics: Adaptation, Physiological; Bias; Biological Evolution; Evolution, Molecular; Models, Genetic; Mutation; Mutation Rate; Phylogeny; Point Mutation; Sequence Homology, Amino Acid
PubMed: 28645195
DOI: 10.1093/molbev/msx180 -
Journal of Visualized Experiments : JoVE Aug 2018Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure...
Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.
Topics: Genetic Variation; Humans; In Situ Hybridization; Point Mutation; RNA
PubMed: 30176002
DOI: 10.3791/58097 -
Folia Neuropathologica 2008It is now widely accepted that many structurally diverse proteins can misfold and cause so-called "conformational diseases", including the most common... (Review)
Review
It is now widely accepted that many structurally diverse proteins can misfold and cause so-called "conformational diseases", including the most common neurodegenerations, Alzheimer's disease and Parkinson's disease. The conversion of largely a-helical or random coil proteins into cross-beta-pleated sheet conformations that form first oligomers and then fibrils underlies these disorders. However, this alpha- to beta-structure transition seems to be a generic propensity of all globular proteins, not only those involved in neurodegenerations, not to mention "prion diseases". Metaphorically, all these neurodegenerations are "infectious" in the sense that misfolded beta-sheeted conformers are formed in a nucleation process in which preformed metastable oligomer acts as a seed (a nucleus) to convert a normal into an abnormal protein. However, in none but transmissible spongiform encephalopathies (TSEs) has infectivity in a microbiological sense ever been observed, and even in TSEs the formation of misfolded protein is not necessarily accompanied by the generation of infectivity de novo. Furthermore, certain "prion diseases" are not TSEs but just "proteinopathies" caused by accumulation of abnormally misfolded PrPd. The presence of a massive amount of PrP-amyloid and no infectivity casts doubts on whether TSEs are really infectious amyloidoses. The misfolding of PrP may yet prove to be an epiphenomenon secondary to infection with a still unknown infectious agent. If, on the other hand, the purely proteinaceous character of the replicating unit of TSE infectivity is ultimately found to be correct, the critical issues become 1) the mechanism by which a misfolded PrP template induces normal protein molecules to adopt the same pathologically misfolded conformation, and 2) the intracellular conditions that are responsible for strain differences in these molecules.
Topics: Amyloid; Humans; Nerve Degeneration; Phenotype; Point Mutation; Prion Diseases; Prions; Terminology as Topic
PubMed: 18587704
DOI: No ID Found -
Bioinformatics (Oxford, England) Nov 2023Accurate prediction of change in protein stability due to point mutations is an attractive goal that remains unachieved. Despite the high interest in this area, little...
MOTIVATION
Accurate prediction of change in protein stability due to point mutations is an attractive goal that remains unachieved. Despite the high interest in this area, little consideration has been given to the transformer architecture, which is dominant in many fields of machine learning.
RESULTS
In this work, we introduce PROSTATA, a predictive model built in a knowledge-transfer fashion on a new curated dataset. PROSTATA demonstrates advantage over existing solutions based on neural networks. We show that the large improvement margin is due to both the architecture of the model and the quality of the new training dataset. This work opens up opportunities to develop new lightweight and accurate models for protein stability assessment.
AVAILABILITY AND IMPLEMENTATION
PROSTATA is available at https://github.com/AIRI-Institute/PROSTATA and https://prostata.airi.net.
Topics: Machine Learning; Neural Networks, Computer; Point Mutation; Protein Stability
PubMed: 37935419
DOI: 10.1093/bioinformatics/btad671 -
Arquivos de Neuro-psiquiatria Sep 2009Rett syndrome (RS) is a severe neurodevelopmental X-linked dominant disorder caused by mutations in the MECP2 gene.
BACKGROUND
Rett syndrome (RS) is a severe neurodevelopmental X-linked dominant disorder caused by mutations in the MECP2 gene.
PURPOSE
To search for point mutations on the MECP2 gene and to establish a correlation between the main point mutations found and the phenotype.
METHOD
Clinical evaluation of 105 patients, following a standard protocol. Detection of point mutations on the MECP2 gene was performed on peripheral blood DNA by sequencing the coding region of the gene.
RESULTS
Classical RS was seen in 68% of the patients. Pathogenic point mutations were found in 64.1% of all patients and in 70.42% of those with the classical phenotype. Four new sequence variations were found, and their nature suggests patogenicity. Genotype-phenotype correlations were performed.
CONCLUSION
Detailed clinical descriptions and identification of the underlying genetic alterations of this Brazilian RS population add to our knowledge of genotype/phenotype correlations, guiding the implementation of mutation searching programs.
Topics: Adolescent; Adult; Brazil; Child; Child, Preschool; DNA; Female; Genetic Association Studies; Humans; Infant; Male; Methyl-CpG-Binding Protein 2; Point Mutation; Polymerase Chain Reaction; Rett Syndrome; Young Adult
PubMed: 19722030
DOI: 10.1590/s0004-282x2009000400001 -
Nucleic Acids Research Jul 2017Here, we report a webserver for the improved SDM, used for predicting the effects of mutations on protein stability. As a pioneering knowledge-based approach, SDM has...
Here, we report a webserver for the improved SDM, used for predicting the effects of mutations on protein stability. As a pioneering knowledge-based approach, SDM has been highlighted as the most appropriate method to use in combination with many other approaches. We have updated the environment-specific amino-acid substitution tables based on the current expanded PDB (a 5-fold increase in information), and introduced new residue-conformation and interaction parameters, including packing density and residue depth. The updated server has been extensively tested using a benchmark containing 2690 point mutations from 132 different protein structures. The revised method correlates well against the hypothetical reverse mutations, better than comparable methods built using machine-learning approaches, highlighting the strength of our knowledge-based approach for identifying stabilising mutations. Given a PDB file (a Protein Data Bank file format containing the 3D coordinates of the protein atoms), and a point mutation, the server calculates the stability difference score between the wildtype and mutant protein. The server is available at http://structure.bioc.cam.ac.uk/sdm2.
Topics: Amino Acid Substitution; Internet; Point Mutation; Protein Stability; Software; Thermodynamics
PubMed: 28525590
DOI: 10.1093/nar/gkx439 -
STAR Protocols Jun 2021CRISPR/Cas9 is an efficient, accurate, and optimizable genome-editing tool. Here, we present a modified CRISPR/Cas9 genome-editing protocol for single nucleotide...
CRISPR/Cas9 is an efficient, accurate, and optimizable genome-editing tool. Here, we present a modified CRISPR/Cas9 genome-editing protocol for single nucleotide mutation in adherent cell lines. The protocol was adapted to focus on ease of use and efficiency. The protocol here describes how to generate a single nucleotide mutation in cultured 22Rv1 cells. We have also used the protocol in other adherent cell types. Thus, the protocol can be applied to assessing the effect of non-coding single nucleotide polymorphisms (SNPs) in a variety of cell types. For complete details on the use and execution of this protocol, please refer to Gao et al. (2018).
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Gene Editing; Humans; MCF-7 Cells; Neoplasms; Point Mutation
PubMed: 33870225
DOI: 10.1016/j.xpro.2021.100419