Methods in enzymology最新文献

Natural products are a rich source of bioactive compounds, which are encoded by the biosynthetic gene clusters (BGCs). Genome mining is an essential strategy for identifying and characterizing BGCs. Targeted genome mining excels in the identification of similar BGCs based on key biosynthetic enzymes across multiple genomes. This chapter details the use of both manual and automated targeted genome mining to identify members of the FK-family BGCs (rapamycin, FK520/506). We describe the process of selecting query proteins, evaluating genomic context, and determining the presence of putative BGCs. Additionally, to streamline the manual process, we used GATOR-GC, a computational tool that identifies similar BGCs using required and optional proteins, performs comparative genomic analysis, deduplicates redundant BGCs, and generates visualizations of gene conservation and BGC diversity. Applying this approach, we showed how to identify FK-family members, both by looking into the cluster conservation diagrams, and the clustered heatmap summarizing all-vs-all BGC comparisons. The methods outlined here can be adapted for mining other natural product families, offering a scalable framework for uncovering novel biosynthetic pathways. Beyond natural product discovery, GATOR-GC provides broader applications for analyzing gene cluster conservation, organization, and evolutionary patterns.

Emerging nascent polypeptides from ribosomes and their protein N-termini have significant impact on protein stability, folding, interaction and subcellular targeting. To profile elongating nascent polypeptides on a proteome-wide scale, here we present a streamlined protocol that combined a biochemical enrichment of puromycin-labeled nascent polypeptides and mass spectrometry-based quantitative proteomics. This chapter includes the detailed protocol for metabolic pulse labeling with puromycin and SILAC amino acids, immunoprecipitation for nascent polypeptides, fractionation and enrichment of N-terminal acetylated peptides as well as MS and data analysis. These protocols are illustrated using HeLa cells treated with cycloheximide, a protein synthesis inhibitor, but can be broadly applied to any cell culture systems, including primary cultures, or any treatments (e.g., drugs).

Artificial metalloenzyme (ArM) design is an attractive approach for deciphering the functional determinants of native enzymes or imparting new functions. Metalloproteins with redox cofactors catalyze critical reactions enabled by optimized primary, secondary, and outer-sphere interactions that facilitate efficient electron transfer. Controlling outer-sphere interactions to tune reactivity remains a challenge. Inspired by the common coordination motifs of copper (Cu) proteins, we have designed artificial Cu proteins (ArCuPs), extensively characterized them, and demonstrated their H₂O₂, O₂, and C-H oxidation reactivity to abiotic substrates. By selectively modifying outer-sphere solvent reorganization energy, we have shown that we can control C-H peroxidation activity. This chapter describes methods to design ArCuPs and their detailed characterization, including electrochemical C-H oxidation, and the procedures for determining reorganization energies using electrochemistry as a readily available laboratory tool.

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.

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.