Reverse engineering the (β/α) ₈ barrel fold

Abstract

The (␤͞␣)8 barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (␤͞␣)8 barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, ␣-helix capping and stop motifs, and residues that pack the interface between ␤-strands and ␣-helices are highly mutable. Conversely, any mutation of residues in the central core of the ␤-barrel, ␤-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that (␤͞␣)8 barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (␤͞␣)8 barrel design.

Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

“…4 Several researchers have demonstrated that the amino acid usage of various natural globular proteins and enzymes can be restricted to 5-13 members without substantial alteration of their structures and biological functions. [5][6][7][8] Riddle et al simplified the sequence of a small b-sheet protein, the SH3 domain, by using phage display selection, and produced two SH3 variants in which 90% of the sequence, excluding the binding region, utilized only five amino acids (Ala, Gly, Glu, Ile, and Lys). 5 Silverman et al generated variants of the prototypical (b/a) 8 barrel enzyme, triosephosphate isomerase (TIM), in which the amino acids at 142 of 182 structural positions were simplified to seven kinds (Ala, Glu, Val, Lys, Phe, Leu, and Gln) by means of in vivo selection for TIM activity.…”

Using diastereopeptides to control metal ion coordination in proteins

“…A common biological structural motif is that of the ␣-helix, and subsequently much de novo design utilizes this scaffold, although ␤-sheets and mixed ␣/␤-constructs have also been investigated (4,(9)(10)(11). Work in our group involves the designed TRI and GRAND peptides based on the heptad repeat, L a K b A c L d E e E f K g , and derivatives thereof (12,13).…”

Simplification of complexity in protein molecular systems by grouping amino acids: a view from physics

“…Therefore, to design the proteins based on a simplified alphabet would provide a way to check the validity of the simplified alphabet. This kind of idea has been realized in many experiments [56][57][58][59][60][61][62][63]. Facing with the helix bundles, de novo designs were realized based on hydrophobic/polar patterns along the protein chains [56,57].…”

“…To reproduce the catalytic activity, more types of amino acids are needed. Through sophisticated designs and selections, active enzymes could be constructed from 9-amino-acid alphabets [61][62][63]. It reflects that there are more demands with further function requirements.…”