title: d-Amino Acid Scan of Two Small Proteins.
abstract: 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.
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Mark D
authors: MD
authors: Simon
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.|Department of Chemistry, Graduate School of Science, Osaka University , 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
authors: Yuta
authors: Y
authors: Maki
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Alexander A
authors: AA
authors: Vinogradov
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Chi
authors: C
authors: Zhang
authors: Department of Chemistry, Tufts University , 62 Talbot Avenue, Medford, Massachusetts 02155, United States.
authors: Hongtao
authors: H
authors: Yu
authors: Department of Chemistry, Tufts University , 62 Talbot Avenue, Medford, Massachusetts 02155, United States.
authors: Yu-Shan
authors: YS
authors: Lin
authors: Department of Chemistry, Graduate School of Science, Osaka University , 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
authors: Yasuhiro
authors: Y
authors: Kajihara
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Bradley L
authors: BL
authors: Pentelute
mesh_terms: D000595:Amino Acid Sequence; D019662:Cucurbitaceae / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D014361:Trypsin Inhibitors / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D014361:Trypsin Inhibitors