Protein prosthesis: β‐peptides as reverse‐turn surrogates

Abstract

The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic b-dipeptide: b 2 -homoalanine-b 3 -homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates.

Building Proficient Enzymes with Foldamer Prostheses

“…The diversity of accessible and stable foldameric secondary structures offers unprecedented opportunities for manipulating protein function. [8] When the conformational propensities of non-natural backbones are highly predictable, as is true of the a/b segments employed here, it may be possible to optimize imperfect prostheses, such as 5-7, through computationally aided laboratory evolution. [18] Ultimately, catalysts that take full advantage of the unique properties of foldamers could enable the installation of novel activities not accessible with natural enzymes.…”

“…A top-down approach in which non-natural oligomer segments are evaluated within an enzymatically competent polypeptide scaffold is a potentially more effective strategy for revealing design criteria that would facilitate the de novo design of active sites. Introducing b-amino acid pairs into a short turn segment of RNase A [8] or swapping an amphiphilic a-helix for a bpeptide surrogate in a chemokine [9] yielded fully functional variants. However, introducing a larger number of artificial building blocks into RNase A reduced activity to below 1 % of that of the wild-type enzyme.…”

Bioengineering strategies to generate artificial protein complexes

“…With the aid of chemical synthesis and EPL, it is even possible to incorporate structural alterations to the backbone of a protein in addition of side‐chain modifications. For instance, Raines and co‐workers replaced a natural reverse turn secondary structure in bovine pancreatic ribonuclease (RNase A) with a “prosthetic” β‐dipeptide that adopts a mimetic reverse‐turn conformation (Arnold et al, ). The resulting protein not only retained wild‐type enzymatic activity, but was also more thermodynamically stable.…”

Building Proficient Enzymes with Foldamer Prostheses

“…Introducing β‐amino acid pairs into a short turn segment of RNase A8 or swapping an amphiphilic α‐helix for a β‐peptide surrogate in a chemokine9 yielded fully functional variants. However, introducing a larger number of artificial building blocks into RNase A reduced activity to below 1 % of that of the wild‐type enzyme 10.…”

“…The guidelines we have identified should facilitate the introduction of nonstandard building blocks into a wide variety of enzymatic scaffolds and thus constitute a first step toward fully unnatural catalysts. The diversity of accessible and stable foldameric secondary structures offers unprecedented opportunities for manipulating protein function 8. When the conformational propensities of non‐natural backbones are highly predictable, as is true of the α/β segments employed here, it may be possible to optimize imperfect prostheses, such as 5 – 7 , through computationally aided laboratory evolution 18.…”