title: Recombinant Preparation, Biochemical Analysis, and Structure Determination of Sirtuin Family Histone/Protein Deacylases.
abstract: Lysine acetylation is long known as a regulatory posttranslational modification of histone proteins and is emerging as a ubiquitous intracellular protein modification. Additional lysine acylations such as succinylation and glutarylation have also been found on histones and other proteins. Acylations are reversibly attached through nonenzymatic acylation mechanisms and the action of protein acyl transferases and protein deacylases (PDACs). Sirtuins are an evolutionary defined class of PDACs and act as metabolic sensors by catalyzing a unique deacylation reaction that requires the cosubstrate NAD(+). Sirtuins are found in all domains of life, and the mammalian sirtuin family comprises seven isoforms in different cellular compartments. They regulate a wide range of cellular targets and functions, such as energy metabolism and stress responses, and they have been implicated in aging processes and aging-related diseases. A large body of functional, biochemical, biophysical, and structural work on isolated sirtuins has provided many important insights that complement the many physiological studies on this enzyme family. They enabled the comprehensive structural and biochemical analysis of sirtuin catalysis, substrate selectivity, and regulation. Here, we describe the recombinant production of sirtuin proteins, with an emphasis on the mammalian isoforms. We then describe their application in activity and binding assays and for crystal structure analysis. We provide protocols for these procedures, and we discuss typical pitfalls in studying this enzyme family and how to avoid them. This information will support further molecular studies on sirtuin mechanisms and functions.
authors.affiliation: Department of Biochemistry, University of Bayreuth, Bayreuth, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Suenkel
authors.affiliation: Department of Biochemistry, University of Bayreuth, Bayreuth, Germany. Electronic address: clemens.steegborn@uni-bayreuth.de.
authors.forename: C
authors.initials: C
authors.lastname: Steegborn
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D044127:Epigenesis, Genetic; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D037761:Sirtuins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D037761:Sirtuins
keywords: Activity assay; Binding assay; Crystal structure; Crystallization; Histone deacetylase; Protein deacetylase; Protein deacylase; Purification; Recombinant expression; Sirtuin