Development of a new protein labeling system to map subunits and domains of macromolecular complexes for electron microscopy.
J Struct Biol
; 201(3): 247-251, 2018 03.
Article
em En
| MEDLINE
| ID: mdl-29170031
ABSTRACT
Several gene fusion technologies have been successfully applied to label particular subunits or domains within macromolecular complexes to enable positional mapping of electron microscopy (EM) density maps, but exogenous fusion of a protein domain into the target polypeptide can cause unwanted structural and functional outcomes. Fab fragments from antibodies can be used as labeling reagents during EM visualization without gene manipulation of the target protein, but this method requires a panel of high-affinity antibodies that recognize a wide variety of epitopes. Linear peptide tags and their anti-tag antibodies can be used but they have a limited mapping ability as their placement is usually limited to the terminal regions of a protein. The PA dodecapeptide epitope tag (GVAMPGAEDDVV), forms a tight ß-turn in the antigen binding pocket of its antibody (NZ-1). This capability allows for insertion of the PA tag into various surface-exposed loops within a multi-domain cell adhesion receptor, αIIbß3 integrin. We confirmed that the purified PA-tagged integrin ectodomain fragments can form a stable complex with NZ-1 Fab. Negative stain EM of the various integrin-NZ-1 complexes revealed that a majority of the particles exhibited a clear density corresponding to the NZ-1 Fab; and the positions of the bound Fab were in good agreement with the predicted location of the inserted PA tag. The high-affinity and insertion-compatibility of the PA tag system allowed us to develop a new EM labeling methodology applicable to proteins for which good antibodies are not available.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Microscopia Eletrônica
/
Proteínas
/
Integrinas
/
Epitopos
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
J Struct Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Japão