posted on 2023-01-12, 22:06authored bySharon
L. Guffy, Surya V. S. R.
K. Pulavarti, Joseph Harrison, Drew Fleming, Thomas Szyperski, Brian Kuhlman
The de novo design of functional proteins
requires
specification of tertiary structure and incorporation of molecular
binding sites. Here, we develop an inside-out design strategy in the
molecular modeling program Rosetta that begins with amino acid side
chains from one or two α-helices making well-defined contacts
with a ligand. A full-sized protein is then built around the ligand
by adding additional helices that promote the formation of a protein
core and allow additional contacts with the ligand. The protocol was
tested by designing 12 zinc-binding proteins, each with 4–5
helices. Four of the designs were folded and bound to zinc with equilibrium
dissociation constants varying between 95 nM and 1.1 μM. The
design with the tightest affinity for zinc, N12, adopts a unique conformation
in the folded state as assessed with nuclear magnetic resonance (NMR)
and the design model closely matches (backbone root-mean-square deviation
(RMSD) < 1 Å) an AlphaFold model of the sequence. Retrospective
analysis with AlphaFold suggests that the sequences of many of the
failed designs did not encode the desired tertiary packing.