1-AminoCyclopropane Carboxylic acid Oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCO have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO.
Characterizing proteins in action requires appropriate biophysical techniques sensitive to protein motions. One of the technique dedicated to monitor protein dynamics is Site-Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR). The main purpose of this chapter is to describe and illustrate the different strategies based on the use of nitroxide spin labels either as reporters or as a means to measure inter-label distances. The complementarity of these different approaches to answer biological questions will be addressed. The objective is also to give non-specialist readers an overview of the recent developments in the field of SDSL-EPR dedicated to the study of protein dynamics. A particular emphasis will be devoted to describe the design and application of new nitroxide spin labels that allow overcoming the limitations of the classical ones.
1‐Aminocyclopropane‐1‐carboxylic oxidase (ACCO) is a non‐heme iron(II)‐containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C‐terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C‐terminal part is reported. Site‐directed spin‐labeling coupled to electron paramagnetic resonance (SDSL‐EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C‐terminal part. Inter‐spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C‐terminal part. This flexibility generates several conformations of the C‐terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.
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