The design and characterization of two proteins with 88% sequence identity but different structure and function

Abstract

To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% sequence identity but different tertiary structures: a 3-␣ helix fold and an ␣/␤ fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the ␣/␤ fold, and the albumin-binding epitope is functional only in the 3-␣ fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.evolution ͉ folding ͉ NMR ͉ protein design ͉ protein structure

A minimal sequence code for switching protein structure and function

“…Proteins were purified using an affinity-cleavage tag system that we developed (16), essentially as described in ref. 3. The system enabled the rapid, standardized purification of mutant proteins, even of low stability.…”

“…In fact, our selection for high- identity alternative folds relied on migration from IgG-binding to albumin-binding function (3). Latent functionality likely exists in nature but is difficult to discern because an alternative function is revealed only in an unknown alternative fold.…”

“…G A and GB variants were cloned into the vector pG58 (16), which encodes an engineered subtilisin prosequence as an N-terminal fusion domain, and the resulting fusion proteins were purified using an affinity-cleavage tag system that we developed (16), essentially as described in ref. 3. A commercial version of the purification system is available through Bio-Rad Laboratories (Profinity eXact Purification System).…”

“…Thus, it seems intuitive that a pathway of single amino acid substitutions would result in a long series of mutants that would be unfolded before enough folding information accumulates to significantly populate an alternative fold. Both natural and engineered examples demonstrate, however, that the sequence space separating 2 proteins with different structures can be quite small (3)(4)(5). To understand this seemingly paradoxical situation, one needs to methodically examine the sequence space separating 2 stable folds.…”

On the efficiency of the Genetic Code after frameshift mutations

“…This allows the side chains to avoid contact with the aqueous environment of the cytoplasm. Due to these many factors, even small changes in the DNA sequence can render a protein useless or completely change its three-dimensional structure (Alexander et al, 2007).…”