title: Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase.
abstract: Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Katja
authors.initials: K
authors.lastname: Conrath
authors.forename: Alice S
authors.initials: AS
authors.lastname: Pereira
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Martins
authors.forename: Cristina G
authors.initials: CG
authors.lastname: Timóteo
authors.forename: Pedro
authors.initials: P
authors.lastname: Tavares
authors.forename: Silvia
authors.initials: S
authors.lastname: Spinelli
authors.forename: Joerg
authors.initials: J
authors.lastname: Kinne
authors.forename: Christophe
authors.initials: C
authors.lastname: Flaudrops
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Isabel
authors.initials: I
authors.lastname: Moura
authors.forename: Jose J G
authors.initials: JJ
authors.lastname: Moura
authors.forename: Mariella
authors.initials: M
authors.lastname: Tegoni
authors.forename: Aline
authors.initials: A
authors.lastname: Desmyter
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D002161:Camelids, New World / Q000276:immunology*; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000276:immunology*; D019151:Peptide Library; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D010088:Oxidoreductases; C034747:nitric-oxide reductase