Methods in enzymology最新文献

Transketolase (TKT), a key rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway, plays a critical role in metabolic processes including nucleotide synthesis and tumorigenesis. Its inhibitors could modulate the enzyme activity and metabolic flux by competitively binding to the cofactor thiamine pyrophosphate (TPP) or allosteric modulatory sites, demonstrating significant potential in drug development for cancer and infectious diseases. In this chapter, we present a systematic evaluation of the binding, affinity and metabolic regulatory activity of a transketolase inhibitor in terms of binding affinity and metabolic regulatory activity. We previously evaluated binding affinity of TKT-inhibitor using two-dimensional (2D) TKT protein biological chromatography, drug affinity responsive target stability assay (DARTS), cellular thermal shift assay (CETSA), surface plasmon resonance analysis (SPR), competitive binding and molecular docking. Moreover, metabolic regulatory activity of a transketolase inhibitor was characterized using spectrophotometric assay and targeted quantitative metabolites analysis, and anti-tumor activity was determined with patient-derived organoids. Notably, several sections of this chapter were originally published in a paper and have been reproduced here for this book.

Transketolases are ubiquitous enzymes that predominantly control the pentose phosphate pathway and are involved in the synthesis of aromatic amino acids, nucleotides and the regulation of oxidative stress. The development of specific inhibitors of human pathogen transketolases could provide a new class of antibiotics. To answer this question, it is necessary to compare human transketolase with those of pathogenic organisms to ensure they are sufficiently different. This chapter presents two protocols for the expression of human and of thirteen transketolases from priority pathogenic organisms, in order to obtain six new experimental structures by X-ray diffraction. Resolution of the electron density maps was performed using in silico models, using detailed protocol for their generation and validation. The experimental structures and models made possible to map and compare for the first time active sites and monomer-monomer interfaces of transketolases. Being at least 50 residues shorter, animal transketolases have evolved differently from those of bacteria, fungi and parasites. The comparison of the monomer-monomer interface also demonstrates that this zone is highly specific to each transketolase, in contrast to their conserved active site. However, in both areas, human transketolase has a significantly higher number of non-covalent bonds than pathogen transketolases, probably to maintain its shorter structure. These observations suggest that pathogen transketolases can be specifically inhibited, particularly targeting the monomer-monomer interface, without affecting human transketolase activity.

Polyamine metabolism in higher eukaryotes is well studied; however, the mechanism of how the polyamines putrescine, spermidine and spermine enter the cell remains unclear. An effective approach to investigate potential players that function in the uptake of polyamines involves using the Drosophila melanogaster imaginal disc assay. Leg imaginal discs dissected from Drosophila melanogaster wandering third star larvae can be assessed for leg development after 18 h of treatment with hormones to induce this process. The protocol described here details how to use genetically manipulated Drosophila melanogaster to test candidate genes involved in the polyamine transport system, how to dissect leg imaginal discs and how to assess the entry of polyamines into the cells of the imaginal disc.

The rise of antibiotic-resistant bacteria poses a critical threat to public health. A key mechanism by which bacteria acquire resistance is through multidrug efflux pumps that expel toxic compounds under antibiotic pressure. Among these, AcrAB-TolC (composed by AcrA, AcrB and TolC, with AcrB belongs to RND family) and MacAB-TolC (composed by MacA, MacB and TolC, with MacB belongs to ABC family) represent two major families of tripartite efflux pump systems in Gram-negative bacteria, each utilizing the same outer membrane channel TolC but differing in their inner membrane components and energization sources. Understanding assembling and functioning mechanism of these pumps requires cellular environment and precise conformational coordination for effective operation. Electron Cryo-tomography (cryoET), in combination with subtomogram averaging, is a unique approach enable direct visualizing macromolecular assemblies within native cellular contexts at subnanometer resolution without any purification, providing critical insights into their in situ architecture, assembly, and function. In this chapter, we present a detailed protocol for the in situ structural characterization of both AcrAB-TolC and MacAB-TolC efflux pumps in Escherichia coli. This unified workflow is broadly applicable to other efflux pumps on other bacterial strains and provides a starting point for studying antibiotic resistance mechanisms.

Multidrug efflux pumps of the Resistance Nodulation-cell Division (RND) superfamily are integral membrane transporters that play a central role in intrinsic and acquired antibiotic resistance in Gram-negative bacteria. Computational approaches have proven invaluable in complementing experimental studies by providing atomistic insight into substrate recognition, transport mechanisms, and inhibitor binding. In this chapter, we provide detailed protocols and tools for most common computational methods applied to RND efflux systems, including homology modelling, molecular docking, all-atom molecular dynamics simulations, and estimation of binding free energy. Each method is presented with practical details on software, input preparation and analysis strategies. Guidelines are included for avoiding common pitfalls and for ensuring reproducibility across computational platforms. Comparisons of the strengths and limitations of these approaches are provided, together with a word of caution on overclaiming results from in silico models without experimental validation. Finally, we discuss the current landscape of computational applications in efflux research illustrating both the opportunities and caveats of these approaches. Together, these methods enable systematic investigation of transporter dynamics, substrate polyspecificity, and inhibition strategies, and can be adapted to other membrane transporters of clinical relevance.

Tripartite efflux pumps are central to multidrug resistance in Gram-negative bacteria, actively extruding antibiotics across the cell envelope. They operate as tripartite complexes spanning the inner and outer membranes, connected by periplasmic adaptors. Here, we describe the in vitro reconstitution of two representative systems into biomimetic environments: the RND-type pump MexAB-OprM from Pseudomonas aeruginosa and the ABC-type pump MacAB-TolC from Escherichia coli. We provide detailed protocols for heterologous expression and purification of individual subunits, followed by their stepwise incorporation into proteoliposomes, nanodiscs, or amphipols. The protocols are adaptable to other Gram-negative multidrug efflux systems and provide a robust platform for dissecting structure-function relationships.

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.