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Publicação
Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase
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Orientador(es)
Resumo(s)
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.
Descrição
Protein Sci. 2009 Mar;18(3):619-28. doi: 10.1002/pro.69.
Palavras-chave
Nitric oxide reductase Camelid antibodies VHH domain SPR Phage display
Contexto Educativo
Citação
Editora
Wiley-Blackwell