J Am Chem Soc. 2016 Sep 21;138(37):12099-111. doi: 10.1021/jacs.6b03765. Epub 2016 Sep 8.
A "D-scan" of two small proteins, the disulfide-rich Ecballium elaterium trypsin inhibitor II (EETI-II) and a minimized Z domain of protein A (Z33), is reported. For each protein, the stereochemistry of one amino acid at a time was inverted to generate a series of diastereomers. In much the same way an alanine scan determines necessary residues for protein function, the D-scan elucidated the critical stereocenters of the 30-residue EETI-II and the 33-residue Z33. The folding properties and activity of each variant were investigated. A total of 24 out of 30 EETI-II D-scan analogues folded to give a three-disulfide product. Of the 24 variants that folded, half were high-affinity trypsin inhibitors, and three were as active as the wild type (WT). Of these 12 active variants, most were substantially less stable to reduction than WT EETI-II (WT first reduction potential -270.0 ± 1.5 mV, WT second reduction potential -307.2 ± 1.1 mV). Similarly, ten Z33 analogues retained high binding affinity to IgG (KD < 250 nM, WT: 24 ± 1 nM) and 12 additional analogues had reduced but appreciable IgG binding affinity (KD between 250 nM and 2.5 μM). As with EETI-II, most Z33 analogues were substantially less stable than the WT (ΔG(H2O, 263 K) = 2.4 ± 1.2 kcal/mol). Collectively, our findings show that the D-scan is powerful new strategy for studying how the stereochemistry of amino acids affects the structure and function of proteins.
Simon MD1, Maki Y1,2, Vinogradov AA1, Zhang C1, Yu H3, Lin YS3, Kajihara Y2, Pentelute BL1.
- 1Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
- 2Department of Chemistry, Graduate School of Science, Osaka University , 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
- 3Department of Chemistry, Tufts University , 62 Talbot Avenue, Medford, Massachusetts 02155, United States.