Article
Received: 20 October 2008; Accepted: 18 December 2008
10.1002/pro.69 About DOI
Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase |
| Katja Conrath 1 2, Alice S. Pereira 3, Carlos E. Martins 3, Cristina G. Timóteo 3, Pedro Tavares 3, Silvia Spinelli 4, Joerg Kinne 5, Christophe Flaudrops 4, Christian Cambillau 4, Serge Muyldermans 1 2, Isabel Moura 3, Jose J. G. Moura 3, Mariella Tegoni 4 *, Aline Desmyter 4 * |
| 1Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium 2Department of Molecular and Cellular Interactions, VIB, Brussels, Belgium 3REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 4Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités of Marseille, Marseille, France 5Central Veterinary Research Laboratory, Dubai, United Arab Emirates |
| email: Mariella Tegoni (mariella.tegoni@afmb.univ-mrs.fr) Aline Desmyter (aline.desmyter@afmb.univ-mrs.fr) |
*Correspondence to Mariella Tegoni, Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités of Marseille, Marseille, France
*Correspondence to Aline Desmyter, Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités of Marseille, Marseille, France
Funded by:
Marseille Nice Génopole® (EU EMEP programme); Grant Number: LSHG-CT2004-504601 Marie-Curie (AST-3D); Grant Number: MEIF-CT-2005-024761 Fundação para a Ciência e a Tecnologia; Grant Number: SFRH/BD/17840/2004, PDTC/QUI/64638/2006
| Keywords |
| nitric oxide reductase camelid antibodies VHH domain SPR phage display |
| Abstract |
| Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N2O. 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 nanobodiesTM). 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. |
Received: 20 October 2008; Accepted: 18 December 2008
| Digital Object Identifier (DOI) |
10.1002/pro.69 About DOI
