Methods in enzymology最新文献

Bacterial efflux pumps are membrane transporters that expel toxic compounds, including antibiotics, from the cell, contributing significantly to multidrug resistance. Among the seven major efflux pump families, transporters from the ATP-binding cassette (ABC) family are primary active systems that use ATP hydrolysis to extrude xenobiotics. Structural studies of these transporters have been advanced by the use of lipid-based reconstitution systems that preserve membrane protein functionality. While nanodiscs have enabled the determination of high-resolution structures, their reconstitution often requires careful optimization. In contrast, peptidisc - a small amphipathic peptide derived from apolipoprotein A-I - may offer a simplified alternative for stabilizing membrane proteins without the need of exogenous lipids. In this chapter, we describe the reconstitution into peptidiscs of PatAB, a type IV ABC transporter from Streptococcus pneumoniae that mediates fluoroquinolone resistance. We explain how mass photometry and size-exclusion chromatography with multi-angle light scattering (SEC-MALS) can be used to evaluate the molecular mass of the transporter in detergent and in peptidisc environments. Additionally, we explain how to reconstitute PatAB into nanodiscs and proteoliposomes, and compared the basal ATPase activity of the transporter in various environments. We highlight the utility of the peptidisc method as a versatile and efficient approach for reconstituting ABC transporters, enabling functional and structural analysis of drug resistance mechanisms.

Chemical modifications are abundant in tRNAs and play essential roles in tRNA biology and human diseases. We recently reported MapID-tRNA-seq that allows identification of chemical modifications in human tRNAs such as m¹A and m³C. MapID-tRNA-seq utilizes an evolved reverse transcriptase (RT-1306) that reads through and generates mutation signatures at m¹A and m³C modifications, which allows robust detection and semi-quantification of m¹A and m³C in human tRNAs. In addition, we developed MapIDs to consolidate the sequence redundancy within the human tRNA genome, with explicit annotations of genetic variance among highly similar tRNA genes. MapIDs help resolve a critical issue of false-positive discoveries of modifications caused by reads misalignment at genetic variance sites. In this chapter, we report detailed protocols for the in-house preparation and characterization of the two enzymes used in MapID-tRNA-seq library preparation (RT-1306 and the demethylase AlkB), tRNA-seq library preparation, and MapID-assisted sequencing analysis, to facilitate application and future development of the MapID-tRNA-seq method.

RNA plays key roles not only as an intermediate between DNA and protein during translation, but also a functional biocatalyst for gene regulation, cell development, environmental interactions and various diseases. In addition to the classical four nucleotides, many chemical modifications located in different positions of the nucleotide further affect and diversify RNA structures and functions. Synthetic RNAs containing these chemical modifications, which are usually made through the well-developed solid phase synthesis, are important toolsets to study RNA biology and develop new therapeutics. This chapter, taking the synthesis of RNAs containing m³C, m⁴C, and m⁴₂C modifications as examples, summarizes the experimental protocols from the synthesis of single nucleoside & phosphoramidite building blocks to the preparation of RNA oligonucleotides using a solid-phase synthesizer, as well as their biophysical and biochemical characterizations, providing a general template for investigating other modified RNAs.

CRISPR-Cas systems use RNA-guided CRISPR-associated (Cas) effectors to neutralize infections in bacteria and archaea. In class 2 CRISPR-Cas systems, Cas9 and Cas12 are single-protein Cas effectors that target double-stranded DNA based on complementarity to the guide RNA before cleaving the target DNA using metal-dependent endonuclease domains. Cas9 and Cas12 proteins can be readily programmed to target any DNA of interest by changing the guiding RNA sequence and have been co-opted for genome editing and other biotechnology purposes. The effect of metal ion concentration is an essential consideration in the physiological role of Cas immunity effectors as well as the biotechnological applications of Cas endonucleases. In this chapter, we describe methods for studying the effect of variable divalent metal ion conditions on the DNA binding and cleavage activities of well-studied Cas9 and Cas12a proteins.

De novo design of artificial metalloproteins offers a powerful approach to dissect and mimic the diverse and exquisite coordination environments found in natural heme proteins. These designed heme proteins seek to replicate the broad array of metabolic, regulatory, and structural functions that hemes perform in biological systems. In this chapter, we present an amenable methodology for the preparation and characterization of de novo-designed heme-binding coiled coils featuring either His- and/or Cys-based axial ligation, which mimic the redox active sites of peroxidases, chloroperoxidases, and cytochrome P450 monooxygenases. The ability to reversibly control heme coordination and spin state through pH variations within a single scaffold provides novel insights into the principles governing catalysis in heme-containing and heme-binding proteins and paves the groundwork for engineering versatile catalysts with tailored reactivity.

According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute an emerging family of natural products, with arising interest in their biosynthetic diversity and therapeutic potentials. Advances in genome sequencing and bioinformatics have significantly accelerated the identification of RiPP biosynthetic gene clusters (BGCs) from genome sequences, however, deciphering the products of these BGCs remains challenging, primarily due to their highly diverse biological origins and elusive genetic regulation machineries. This chapter describes the use of pathway reconstruction approaches for exploring the biosynthetic potential of cryptic RiPP BGCs. Specifically, a plug-and-play pathway refactoring workflow is described, which can effectively rewire the underlying regulatory systems in target BGCs, ensuring their expression in genetically tractable organisms. In addition, the Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), a method capable of cloning large DNA fragments into selected expression vectors, is provided as an alternative way for investigating BGCs with intricate gene arrangements. Due to their high efficiency and robustness, these methods would be of interest to those working on the genome mining of RiPPs, as well as other families of natural products.

Natural products are diverse compounds made by many organisms, though bacteria, fungi, and plants are particularly prolific producers. While they have a range of biological roles, bioactive natural products have long been of interest as drug candidates. With the advent of accessible genome mining tools like antiSMASH, it is possible to search through genomes and metagenomes, identifying genes associated with natural product production and even predicting potential structures for experimentally uncharacterized compounds. However, most genome mining tools rely on similarity to previously characterized natural product pathways, and so they can fail to detect unusual or novel pathways and pathways that rely on "hypothetical proteins" for key biosynthetic steps. This is unfortunate, because natural products from new classes or with potentially divergent scaffolds are of particular interest in efforts to identify compounds with antibiotic and anticancer activity. This chapter will document some of the approaches that can be used to explore and develop biosynthetic hypotheses for these challenging-to-detect natural product pathways.

The N-terminus of a protein has an important regulatory impact on its in vivo stability and half-life. Proteins destined to chloroplasts and mitochondria are synthesized as precursor proteins in the cytosol with an N-terminal peptide sequence that ensures their correct targeting. During their cytosolic passage, precursor proteins are exposed to the cytosolic protein degradation machinery, hence, their N-termini must comply with regulatory processes for proteolytic degradation in the cytosol. We present here a method to determine the identity and modification state of plastid precursor protein N-termini in the cytosol by combining protoplast protein import assays with targeted mass spectrometry by means of parallel reaction monitoring (PRM). This method requires a hypothesis on potential modifications at the protein N-terminus such as methionine removal or N-terminal acetylation, that is decoded into an inclusion mass list to guide mass spectrometric data acquisition to specific peptides. This type of approach largely eliminates the stochastic nature of MS acquisition allowing different modifications to be tested as alternative hypotheses. Using Skyline, a quantitative assessment of different N-terminal modifications can be performed. We have used this method to determine the modification state of a model precursor protein RNP29 in two different genotypic backgrounds, but our workflow is easily expandable to different precursors.