title: Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge.
abstract: Many recombinant proteins carry an oligohistidine (His(X))-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion-nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.
authors.affiliation: Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.
authors.forename: Lance M
authors.initials: LM
authors.lastname: Hellman
authors.forename: Chunxia
authors.initials: C
authors.lastname: Zhao
authors.forename: Manana
authors.initials: M
authors.lastname: Melikishvili
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Tao
authors.forename: James E
authors.initials: JE
authors.lastname: Hopper
authors.forename: Sidney W
authors.initials: SW
authors.lastname: Whiteheart
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fried
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D006639:Histidine