A hybrid sequence approach to the paracelsus challenge

Abstract

Inspired by the Paracelsus Challenge of Rose and Creamer (Proteins 19: 1-3, 1994), we have designed a protein sequence that is 50% identical to an all-helical protein but is intended to fold into a largely beta-sheet structure. Rather than attempt a de novo design, our strategy was to construct a hybrid sequence based on a helical "parent" protein (434 Cro) and a "target" protein with the desired fold (the B1 domain of protein G). The hybrid sequence (Crotein-G) is 50% identical to 434 Cro but is also 62% identical to the B1 domain of protein G. We also created a variant of Crotein-G (ZCrotein-G) that contains a potential His3Cys1 zinc binding site. At low protein concentrations and in the presence of 20% 2,2,2-trifluoroethanol (TFE) (v/v), the circular dichroism spectra of the designed proteins are distinct from that of 434 Cro and similar to that of the B1 domain of protein G. However, the proteins fail to denature in a cooperative manner. Furthermore, aggregation occurs at moderate protein concentrations or in the absence of TFE. Addition of zinc to ZCrotein-G does not promote structure formation. In summary, 434 Cro has been altered to something that may resemble the B1 domain of protein G, but the protein does not adopt a native structure.

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

“…The other 49 positions influence the 3-␣ to U and the ␣/␤ to U equilibria, but they can be tolerated in both folds and therefore provide a relatively neutral sequence background in which to observe an underlying fold-specific folding code. Clearly the complementary set of mutations in each member of the pair have to be very carefully selected to achieve this level of identity, but previous studies indicate that this result is probably not unique to this pair of proteins (17)(18)(19)(20)(21).…”

“…The positions of nonidentity in the heteromorphic pair of highest identity constitute an essential fold-specific folding code parsed from the overall stability code. This basic approach has been taken in a number of previous studies (17)(18)(19)(20)(21). We have been able to build on the previous work to create heteromorphic pairs of higher identity and higher stability while preserving biological function.…”

Interplay Between Metal Binding and cis/trans Isomerization in Legume Lectins: Structural and Thermodynamic Study of P. angolensis Lectin

“…This was most strikingly shown by the completion of the Paracelsus challenge, where a polypeptide was designed that switches from one fold to another by substitution of less than 50% of its amino acid residues. [42][43][44] Other data corroborating this idea is the occasional detection of intramolecular switches: point mutations that change the protein fold locally, mediate a transition between α-helix and β-strand conformations or change its oligomerization state. 45,46 The correct identification of such switches is highly relevant for homology modeling, fold recognition and protein design.…”

NMR structures of two designed proteins with high sequence identity but different fold and function

“…The nonidentities between these two amino acid sequences would then be responsible for coding one fold topology over the other and, thus, represent a fold-specific folding code. Several groups responded to this challenge (6)(7)(8)(9)(10) and the most successful of these studies resulted in a protein pair that had 80% sequence identity (11). Some of these proteins were characterized by circular dichroism (CD) spectroscopy under acidic conditions but had a propensity to aggregate near neutral pH.…”