Regulation of Tamavidin–Biotin Affinity by the C-Terminal of the Native Protein

Abstract

Tamavidin 2 is a biotin-binding protein of the avidin-like family, discovered in Tamogitake mushrooms, that has found a number of uses in biotechnology. Unlike avidin or streptavidin, it can be expressed in Escherichia coli as a soluble protein, making it a good partner in chimaeric proteins. Surprisingly, we found that the full-length protein purified from E. coli has a much lower affinity for biotin than that of avidin or streptavidin. In fact, a micromolar Kd was determined from fluorescence measurements, in contrast to the femtomolar Kd of avidin. Interestingly, treatments with trypsin or denaturing agents resulted in a large increase in the affinity. Modeling of the structure suggested that the C-terminal five amino acids obscured the biotin-binding site in the complete protein. Indeed, alterations of these five amino acids, either by truncation or individual mutation to alanine, resulted in a thousand-fold or greater increase in affinity for biotin. A truncated tamavidin was much more effective in forming complexes with biotinylated target proteins and as a green fluorescent protein (GFP) conjugate in binding assays. The inhibited state of the full-length protein and its activation by proteolysis suggest that tamavidin functions as a defensive protein to discourage predation.