Towards meeting the paracelsus challenge: The design, synthesis, and characterization of paracelsin-43, an α-helical protein with over 50% sequence identity to an all-β protein

Jones, David T.; Moody, Claire M.; Uppenbrink, Julia; Viles, John H.; Doyle, Paul M.; Harris, Christopher; Pearl, Laurence H.; Sadler, Peter J.; Thornton, Janet M.

doi:10.1002/(sici)1097-0134(199604)24:4<502::aid-prot9>3.0.co;2-f

Cited by 43 publications

(29 citation statements)

References 36 publications

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Alexander

Chen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

176

192

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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

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Alexander

Chen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

176

192

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show abstract

“…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.…”

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confidence: 99%

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

Chen²,

Alexander³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

106

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How protein sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal sequence identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, GA88 and GB88, with 88% sequence identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963-11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the sequence identity level from 16 to 88%, GA88 and GB88 maintain their distinct wild-type 3-␣ and ␣/␤ folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high sequence identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-␣ and ␣/␤ conformations. Further mutation of a subset of these nonidentities, guided by the GA88 and GB88 structures, leads to proteins with even higher levels of sequence identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.conformational switching ͉ evolution ͉ folding U nderstanding the relationship between protein sequence and 3D structure (1) remains the fundamental unresolved problem in structural biology. There are several reasons why the protein folding problem is so difficult. The large number of conformations available to even a short polypeptide chain makes it difficult to calculate which conformation is most preferred. Also, amino acids in a polypeptide sequence contribute to different extents in coding for a particular fold (2-4). Mutations at some positions will have negligible effect on protein stability whereas other residues cannot be altered without resulting in complete unfolding. In this sense, most natural folds can be considered to be only marginally stable. The combination of these factors results in the general observation that many sequences with little or no discernible homology can frequently have the same overall fold, making prediction of 3D structure from sequence highly problematic in such cases.A different way of looking at this problem was posed by Rose and Creamer (5). They suggested that one could gain insights into the folding code by determining the minimum number of amino acids required to specify one fold over another. The basic idea was to design a pair of proteins with maximal sequence identity but retaining their different wild-type folds. The nonidentities between these two amino acid sequences would then be responsible for coding one fold top...

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“…If a domain eventually assumes a new fold by amino acid substitutions, there must be a point where enough mutations have accumulated to switch from one fold to another. Determining this point (i.e., the number of amino acid changes necessary) has been a challenge for some time now, resulting, for example, in the Paracelsus challenge, which called for a conversion between two protein folds while retaining 50% of the original amino acid composition (Rose and Creamer, 1994;Jones et al, 1996). This goal was achieved [with the Janus protein (Dalal et al, 1997)] and even exceeded recently by a pair of proteins sharing 88% of their amino acids but assuming different folds (Alexander et al, 2007).…”

Section: Structural Promiscuity Of Proteinsmentioning

confidence: 99%

Evolution AfterandBefore Gene Duplication?

Sikosek

Bornberg‐Bauer

2010

Evolution After Gene Duplication

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Towards meeting the paracelsus challenge: The design, synthesis, and characterization of paracelsin-43, an α-helical protein with over 50% sequence identity to an all-β protein

Cited by 43 publications

References 36 publications

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

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

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

Evolution AfterandBefore Gene Duplication?

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