Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.
Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.
Helppolainen
Satu H
SH
Institute of Medical Technology, University of Tampere, FI-33014 Tampere, Finland.
Nurminen
Kirsi P
KP
Määttä
Juha A E
JA
Halling
Katrin K
KK
Slotte
J Peter
JP
Huhtala
Tuulia
T
Liimatainen
Timo
T
Ylä-Herttuala
Seppo
S
Airenne
Kari J
KJ
Närvänen
Ale
A
Jänis
Janne
J
Vainiotalo
Pirjo
P
Valjakka
Jarkko
J
Kulomaa
Markku S
MS
Nordlund
Henri R
HR
D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012231:Rhizobium / Q000378:metabolism*
D001426:Bacterial Proteins; C535197:rhizavidin, Rhizobium etli; D001360:Avidin