Abstract
Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10−15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.
Highlights
Due to the highly stable beta-barrel structure, high resistance to a wide range of temperature and pH changes, rather small size and, especially, the extraordinary tight binding to a small molecule D-biotin, avidins (Avds) are exploited in a number of practical life science applications and biotechnological assays
The X-ray structure of sbAvd-2 (I117Y) in a putative complex with progesterone [PDB:5LUR] was determined at 2.80 Å resolution. This antidin was co-crystallized with progesterone and the two binding sites of the asymmetric unit have clear blobs of electron density, most likely representing progesterone molecules even though the density at the binding sites was worse, or less interpretable, than for most regions of the polypeptide chains (Fig 2)
To our recently published apo structure of sbAvd-2 (I117Y) [PDB:4U46], the overall fold of the complex structure closely resembled that of chicken Avd [19]
Summary
Due to the highly stable beta-barrel structure, high resistance to a wide range of temperature and pH changes, rather small size (around 60 kDa for a tetramer) and, especially, the extraordinary tight binding to a small molecule D-biotin, avidins (Avds) are exploited in a number of practical life science applications and biotechnological assays. These include techniques for imaging, purification, labeling, targeting and detection [1,2]. Fimlab and the other funders had no influence in the study design, data collection and analysis, decision to publish or preparation of the manuscript
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