Methods in enzymology最新文献

Non-heme iron enzymes play key roles in antibiotic, neurotransmitter, and natural product biosynthesis, DNA repair, hypoxia regulation, and disease states. These enzymes had been refractory to traditional bioinorganic spectroscopic methods. Thus, we developed variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy to experimentally define the excited and ground ligand field states of non-heme ferrous enzymes (Solomon et al., 1995). This method provides detailed geometric and electronic structure insight and thus enables a molecular level understanding of catalytic mechanisms. Application of this method across the five classes of non-heme ferrous enzymes has defined that a general mechanistic strategy is utilized where O₂ activation is controlled to occur only in the presence of all cosubstrates.

Methyl-coenzyme M reductase (MCR) is the key enzyme in pathways for the formation and anaerobic oxidation of methane. As methane is a potent greenhouse gas and biofuel, investigations of MCR catalysis and maturation are of interest for the development of both methanogenesis inhibitors and natural gas conversion strategies. The activity of MCR is dependent on a unique, nickel-containing coenzyme F430, the most highly reduced tetrapyrrole found in nature. Coenzyme F430 is biosynthesized from sirohydrochlorin in four steps catalyzed by the CfbABCDE enzymes. Here, methods for the expression and purification of the coenzyme F430 biosynthesis enzymes are described along with conditions for the synthesis and purification of biosynthetic intermediates on the milligram scale from commercially available porphobilinogen.

Eukaryotic cells require energy to perform diverse cellular functions critical for survival. Mitochondria are multifunctional organelles that generate energy in the form of Adenosine triphosphate by oxidative phosphorylation, emphasizing their importance to eukaryotic cell viability. The ability of mitochondria to consume oxygen for respiration is a key parameter in assessing mitochondrial health. Therefore, developing new techniques to monitor mitochondrial respiration are crucial for advancing our understanding of organelle functioning. Recently, Seahorse technology has emerged as a valuable tool to analyze various aspects of mitochondrial bioenergetics. Although the Seahorse assay is well established in adherent cell lines and other model organisms, it remains challenging to employ it efficiently in yeast, a powerful genetic system for studying mitochondrial biology. In this chapter, we provide a comprehensive methodology for assessing oxygen consumption rate in baker's yeast using Seahorse.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1, APEX1, REF1, HAP1) is an abasic site-specific endonuclease holding critical roles in numerous biological functions including base excision repair, the DNA damage response, redox regulation of transcription factors, RNA processing, and gene regulation. Pathologically, APE1 expression and function is linked with numerous human diseases including cancer, highlighting the importance of sensitive and quantitative assays to measure APE1 activity. Here, we summarize biochemical and biological roles for APE1 and expand on the discovery of APE1 inhibitors. Finally, we highlight the development of assays to monitor APE1 activity, detailing a recently improved and stabilized DNA Repair Molecular Beacon assay to analyze APE1 activity. The assay is amenable to analysis of purified protein, to measure changes in APE1 activity in cell lysates, to monitor human patient samples for defects in APE1 function, or the cellular and biochemical response to APE1 inhibitors.

In this chapter we survey prediction tools and computational methods for the prediction of amino acid sequence elements which target proteins to the mitochondria. We will primarily focus on the prediction of N-terminal mitochondrial targeting signals (MTSs) and their N-terminal cleavage sites by mitochondrial peptidases. We first give practical details useful for using and installing some prediction tools. Then we describe procedures for preparing datasets of MTS containing proteins for statistical analysis or development of new prediction methods. Following that we lightly survey some of the computational techniques used by prediction tools. Finally, after discussing some caveats regarding the reliability of such methods to predict the effects of mutations on MTS function; we close with a discussion of possible future directions of computer prediction methods related to mitochondrial proteins.

The isolation of intact and functional mitochondria is a powerful approach to characterize and study this organelle. The classical biochemical method of differential centrifugation is routinely used to isolate mitochondria. This method has several advantages, such as a high yield and easy adaptability. The isolated mitochondria are physiologically active and can be used for a variety of follow-up experiments, for example protein import and respiration measurements. Here, we describe the procedure to purify mitochondria from the budding yeast Saccharomyces cerevisiae. In addition, two approaches are introduced to assess the quality of isolated mitochondria, by limited proteinase K digestion or measurement of the membrane potential.

The translocase of the mitochondrial inner membrane (TIM23) complex mediates the import and membrane insertion of presequence-carrying mitochondrial proteins. It is experimentally challenging to determine whether the segment of the polypeptide is imported to the matrix or inserted into the inner membrane. Utilizing the unique topogenesis of Mgm1p, a versatile experimental approach to study the TIM23-mediated membrane insertion is developed and described in this chapter. This method combines a simple SDS-gel based assay with the quantification of the relative fractions of membrane inserted and non-inserted products, enabling the quantitative measurement of the membrane insertion efficiencies of a transmembrane segment into the mitochondrial inner membrane.

The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient "cloggers" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.

The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets.

The mitochondrial import machinery is regulated by several protein kinases that phosphorylate key components. This allows an adjustment of the protein flux to changing cellular demands and allow a dynamic organellar proteome. PhosTag electrophoresis has been proven as highly valuably tool to study these signalling machanisms at the import machinery.