Towards the Design of Metamorphic Proteins using Ensemble-Based Energetic Information

Abstract

The discovery of natural and engineered proteins that adopt more than one functionally relevant structure represents an emerging paradigm shift in the field of protein folding and stability. This work explores a preliminary approach to understanding how a single amino acid sequence may adopt more than one stable structure. Key to the approach is information derived from a previously reported (and experimentally validated) statistical mechanical ensemble description of globular protein thermodynamics. Such energetic information is demonstrated to correctly match and align amino acid sequences to the corresponding known structures in large databases. As a first step, an algorithm was developed to generate novel amino acid sequences energetically compatible with a single target structure, the SH3 domain. Information about both the native and denatured state energetics of the target was taken into account, as we hypothesize that the denatured state in particular cryptically encodes necessary negative design information. These designed sequences indeed demonstrated primary, secondary, and tertiary structure properties similar to known SH3 domains in silico. Experimental characterization of one designed sequence revealed reversible cooperative two-state denaturation, consistent with the expected biophysical properties of an SH3 domain. Encouraged by these results, we are proceeding to discover naturally occurring sequences that are energetically compatible with more than one structure. Initial analysis suggests that natural sequences exhibit a surprisingly large range of compatibility with non-self structures, with some so-called “promiscuous” sequences potentially compatible with many structures. Statistically enriched regions of sequence containing glycine, matching with low-stability (e.g. turn) regions in the non-self structure, appears to be one general mechanism mediating this energetic “promiscuity”. Such insight, only possible because of our unique thermodynamic approach to protein design, may become part of a future experimental strategy to engineer metamorphic proteins.