Methods in enzymology最新文献

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.

Mitochondria contain about 1000 different proteins, only a handful of which are encoded in the mitochondrial genome. The remaining c. 99% of mitochondrial proteins are encoded in the nuclear genome, synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals and are subsequently imported into the organelle. Mitochondrial targeting signals are very diverse and mitochondria therefore also have a number of very sophisticated molecular machines that recognize, import and sort mitochondrial precursor proteins to the different mitochondrial subcompartments. The ability to synthesize mitochondrial precursor proteins in vitro and subsequently import them into isolated mitochondria has revolutionized our understanding of mitochondrial protein import pathways. Here, we describe the basic protocol for synthesis of mitochondrial precursor proteins in vitro and their subsequent import into isolated mitochondria from yeast Saccharomyces cerevisiae, the method which was used to elucidate and characterize the vast majority of mitochondrial protein import pathways.

Precise protein localization is essential for normal cellular functions. However, recent studies have revealed that protein targeting is error-prone, and tail-anchored proteins mistargeted to mitochondria are transferred to the endoplasmic reticulum (ER) by an ATPase Msp1 (yeast)/ATAD1 (human) in the mitochondrial outer membrane for further quality examination in the ER to determine their fate, degradation or re-targeting. Analysis of the inter-organelle transfer of proteins requires a combination of time-lapse fluorescence microscopy and a system to achieve regulation of the protein levels of both transfer substrates and factors regulating the transfer in a coordinated manner at precise timing. This can be achieved by using a promoter switch for expression and acute depletion of involved factors through the degron-based proteasome system. In this chapter, we will describe methods to analyze inter-organelle protein transfer by fluorescence microscope within living yeast cells, by using the example of Msp1-mediated transfer of mistargeted proteins from mitochondria to the ER.

The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are "born" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.

Climate change due to anthropomorphic emissions will increase global temperature by at least 1.5 °C by the year 2030. One strategy to reduce the severity of the effects of climate change is to sequester carbon dioxide via natural biochemical cycles. Carbon monoxide dehydrogenase (CODH) has the remarkable ability to catalyze the reversible reduction of CO₂ to CO without an overpotential and without reducing protons. It also is a key enzyme in the Wood-Ljungdahl pathway (WLP), which is the only known anaerobic carbon fixation pathway and fixes 10 % of carbon on earth every year. Characterization of this pathway is crucial because it may enable tools to mitigate climate change by using CO₂ to produce biofuels, chemical feedstocks, and polymers. In the WLP, CODH associates with Acetyl-Coenzyme A synthase (ACS), which catalyzes the condensation of CO from CODH, a methyl group from a B₁₂-dependent methyltransferase, and CoA to form acetyl-CoA. In this complex, CO is shuttled through a 138 Å gas tunnel between the two enzymes. One valuable model for studying the CODH component of CODH/ACS is CODH-II from Carboxydothermus hydrogenoformans because it is stand-alone and is conducive to recombinant expression. Here we describe a detailed protocol for producing high-activity CODH-II in E. coli.

Guanidine metabolism has been an overlooked aspect of the global nitrogen cycle until RNA sensors (riboswitches) were discovered in bacteria that bind the nitrogen-rich compound. The associated genes were initially proposed to detoxify guanidine from the cells. We were intrigued by a genetic organization where the guanidine riboswitch is located upstream of an operon comprising a carboxylase, two putative hydrolases, and an assigned allophanate hydrolase. An ABC transporter is located on the same operon with a periplasmic binding domain that is indicative of an importer. Therefore, we hypothesized that certain bacteria actively import guanidine and assimilate the nitrogen. To test this hypothesis, we searched for bacteria that were able to assimilate guanidine. We isolated three enterobacteria (Raoultella terrigena str. JH01, Erwinia rhapontici str. JH02 and Klebsiella michiganensis str. JH07) that utilize guanidine efficiently as a nitrogen source. Proteome analyses demonstrate that the expression of the guanidine riboswitch-associated carboxylase, in conjunction with associated hydrolases and transport genes, is markedly elevated in the presence of guanidine. Subsequent analysis of the carboxylases that are homologous to urea carboxylase confirmed the substrate preference of guanidine over urea. This chapter outlines a procedure for the isolation of guanidine-assimilating bacteria and the analysis of their proteome to identify enzymes responsible for guanidine degradation. Finally, an assay for the characterization of the endogenous guanidine carboxylases in comparison with the endogenous urea carboxylase from E. rhapontici is described.

Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells. This is seemingly due to the lack of experimental strategies to perturb the lipid composition in one leaflet and quantitative techniques to evaluate the biophysical properties of the opposite leaflet. The existing strategies often dependent on immobilization and clustering a component in one of the leaflets and technically demanding biophysical tools to evaluate the effects on the opposing leaflet. In the recent years, the London group developed a simple but elegant method, namely methyl-alpha-cyclodextrin catalyzed lipid exchange (LEX), to efficiently exchange outer leaflet lipids with an exogenous lipid of choice. Here, we adopted this method to perturb outer leaflet lipid composition. The corresponding changes in the inner leaflet is evaluated by comparing the diffusion of lipid probes localized in this leaflet in unperturbed and perturbed conditions. We employed highly multiplexed imaging fluorescence correlation spectroscopy (ImFCS), realized in a commercially available or home-built total internal reflection fluorescence microsocope equipped with a fast and sensitive camera, to determine diffusion coefficient of the lipid probes. Using the combination of LEX and ImFCS, we directly demonstrate lipid-based transbilayer coupling that does not require immobilization of membrane components in live mast cells in resting conditions. Overall, we present a relatively straightforward experimental strategy to evaluate transbilayer coupling quantitively in live cells.

Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.

This chapter describes additions to the DIALS software package for processing serial still-shot crystallographic data, and the implementation of a pipeline, xia2.ssx, for processing and merging serial crystallography data using DIALS programs. To integrate partial still-shot diffraction data, a 3D gaussian profile model was developed that can describe anisotropic spot shapes. This model is optimised by maximum likelihood methods using the pixel-intensity distributions of strong diffraction spots, enabling simultaneous refinement of the profile model and Ewald-sphere offsets. We demonstrate the processing of an example SSX dataset where the improved partiality estimates lead to better model statistics compared with post-refined isotropic models. We also demonstrate some of the workflows available for merging SSX data, including processing time/dose resolved data series, where data can be separated at the point of merging after scaling and discuss the program outputs used to investigate the data throughout the pipeline.