Methods in enzymology最新文献

Single-particle cryo-electron microscopy (cryo-EM) has become a very powerful technique in the field of membrane protein structural biology. Historically, protein structure determination requires homogenous and pure samples, and sample heterogeneity often hampered the progress of drug design and development, especially those targeting membrane proteins and their complexes. With the rapid development of the instrumentation, software and methodologies of cryo-EM, it is now feasible to obtain high-resolution cryo-EM structural information of membrane proteins, from both pure/homogeneous and impure/heterogeneous samples. Here, we present our current protocols and methodologies for this structural technique. Case studies show step-by-step how we used this cryo-EM methodology to elucidate the structure and assembly of the important mycobacterial membrane protein large (MmpL) family of transporters. It is our intention to encourage more researchers to employ a variety of structural, biophysical and biochemical methodologies to continue to study critical membrane proteins for the development of novel therapeutic strategies to combat human diseases.

Cyclotides are a unique class of head-to-tail cyclic peptides with exceptional stability, making them promising scaffolds for therapeutic and agrochemical applications. Their biosynthesis in plants involves asparaginyl endopeptidases (AEPs), which catalyze backbone cyclization through transpeptidation. This chapter presents a detailed chemoenzymatic method for producing cyclotides using AEP-mediated cyclization, focusing on the model cyclotide kalata B1. The method leverages the high efficiency and specificity of AEPs, enabling cyclization of folded substrates without the need for protecting groups or harsh chemical reagents. This approach is scalable and adaptable to other cyclotides and bioactive peptides, offering a robust platform for generating stable, cyclic peptides with enhanced therapeutic potential.

Bioactive peptides (BPs) are promising therapeutic agents due to their high selectivity and low toxicity. However, their clinical potential is often limited by rapid enzymatic degradation and poor pharmacokinetic profiles. N-methylation of the peptide backbone has emerged as an effective strategy to improve proteolytic stability, membrane permeability, and conformational control by limiting intramolecular hydrogen bonding and restricting structural flexibility. In this chapter, we present a simplified and cost-effective solid-phase peptide synthesis (SPPS) protocol for the preparation of N-methylated peptides and lipopeptides. The method employs Fmoc chemistry, DIC/HOBt coupling, and ChemMatrix Rink Amide resin under manual conditions, without requiring specialized instrumentation. This protocol enables the efficient incorporation of one or more N-methylated residues and is suitable for the development of protease-resistant analogs. It provides a practical tool for researchers aiming to enhance the metabolic stability and pharmacological potential of therapeutic peptide candidates.

Although canonical protein design has benefited from machine learning methods trained on databases of protein sequences and structures, synthetic heteropolymer design still relies heavily on physics-based methods. The Rosetta software, which provides diverse physics-based methods for designing sequences, exploring conformations, docking molecules, and performing analysis, has proven invaluable to this field. Nevertheless, Rosetta's aging architecture, monolithic structure, non-open source code, and steep development learning curve are beginning to hinder new methods development. Here, we introduce the Masala software suite, a free, open-source set of C++ libraries intended to extend Rosetta and other software, and ultimately to be a successor to Rosetta. Masala is structured for modern computing hardware, and its build system automates the creation of application programming interface (API) layers, permitting Masala's use as an extension library for existing software, including Rosetta. Masala features modular architecture in which it is easy for novice developers to add new plugin modules, which can be independently compiled and loaded at runtime, extending functionality of software linking Masala without source code alteration. Here, we describe implementation of Masala modules that accelerate protein and synthetic peptide design. We describe the implementation of Masala real-valued local optimizers and cost function network optimizers that can be used as drop-in replacements for Rosetta's minimizer and packer when designing heteropolymers. We explore design-centric guidance terms for promoting desirable features, such as hydrogen bond networks, or discouraging undesirable features, such as unsatisfied buried hydrogen bond donors and acceptors, which we have re-implemented far more efficiently in Masala, providing up to two orders of magnitude of speedup in benchmarks. Finally, we discuss development goals for future versions of Masala.

Transketolases (TKs) are important C-C bond forming enzymes that in vivo transfer a two carbon ketol unit to the acceptors d-ribose-5-phosphate or d-erythrose-4-phosphate. There is significant interest in biocatalytic applications where frequently the donor β-hydroxypyruvic acid is used. In recent years there has been interest in the discovery of new TKs with unique or robust properties that are an excellent starting point for mutagenesis, or that are able to accept new acceptors or donors. Similarly, TK mutagenesis has led to TKs with alternative substrate profiles. In this chapter, firstly an overview of the substrates accepted by TKs is briefly summarized. Then, metagenomic strategies for the discovery of unique TKs and how this approach has developed with an early example, and a more recent study on the discovery of 'split'-TKs, are described with methods. Finally, enzyme evolution methods and approaches to develop a wide range of TKs with modified substrate acceptance and improved stabilities are detailed.

Small non-coding RNAs, including microRNAs (miRNAs) and tRNA fragments (tRFs), play critical roles in gene regulation across diverse biological contexts. Although miRNAs and tRFs are traditionally viewed as cytoplasmic effectors, recent studies suggest they may also adopt functions within the nucleus. However, accurately mapping the subcellular localization of small RNAs remains technically challenging due to inherent biases in RNA yield between the nuclear and cytoplasmic compartments, as well as the presence of base-pair-disrupting RNA modifications. Here, we present a method for small RNA sequencing in example glioblastoma (GBM) cell lines that enables accurate subcellular localization by integrating defined synthetic spike-in controls and Induro-RT, a highly processive reverse transcriptase capable of reading through modified nucleotides. Spike-in controls correct for input disparities across compartments, while Induro-RT allows for the transcription of modified small RNAs, which are often overlooked by conventional reverse transcriptases. This approach enables unbiased detection of both canonical and modified small RNAs, providing a more accurate and comprehensive view of miRNA and tRF distribution between the nucleus and cytoplasm.

As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.

Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.

By employing site-directed RNA editing (SDRE) to restore point-mutated RNA molecules, it is possible to change gene-encoded information and synthesize proteins with different functionality from a single gene. Thymine (T) to cytosine (C) point mutations cause various genetic disorders, and when they occur in protein-coding regions, C-to-uridine (U) RNA changes can lead to non-synonymous alterations. By joining the deaminase domain of apolipoprotein B messenger RNA (mRNA) editing catalytic polypeptide 1 (APOBEC1) with a guide RNA (gRNA) complementary to a target mRNA, we created an artificial RNA editase. We used an mRNA encoding blue fluorescent protein (BFP), obtained from the green fluorescent protein (GFP) gene through the introduction of a T > C mutation, as our target RNA. In a proof of principle experiment, we reverted the T > C mutation at the RNA level using our APOBEC1 site-directed RNA editing system, recovering GFP signal. Sanger sequencing of cDNA from transfected cells and polymerase chain reaction-restriction length polymorphism analysis validated this result, indicating an editing of approximately 21 %. Our successful development of an artificial RNA editing system using the deaminase APOBEC1, in conjunction with the MS2 system, may lead to the development of treatments for genetic diseases based on the restoration of specific types of wild type sequences at the mRNA level.

Transfer RNA-derived RNAs (tDRs) have emerged as important regulatory molecules found across all three domains of life. Despite their discovery over four decades ago, their biological significance has only recently begun to be elucidated. However, studying bacterial tDRs poses challenges due to technical limitations in assessing their in vivo functionality. To address this, we established a novel approach utilizing a self-cleaving Twister ribozyme to express tDRs in Escherichia coli. Specifically, we employed the type P1 Sva1-1 Twister ribozyme, to generate tDRs with genuine 3' ends. Our method involves the inducible expression of tDRs by incorporating the desired tDR sequence into a plasmid construct downstream of two lac operators and upstream of the Twister ribozyme. Upon induction with IPTG and transcription of the construct, the Twister ribozyme undergoes self-cleavage, thus producing tDRs with defined 3' ends. As a proof of principle, we demonstrated the in vivo application of our novel method by expressing and analyzing two stress-induced tRNA halves in E. coli. Overall, our method offers a valuable tool for studying tDRs in bacteria to shed light on their regulatory roles in cellular processes.