Oligomerization of Uniquely Folded Mini-Protein Motifs:  Development of a Homotrimeric ββα Peptide

Mezo, Adam R.; Cheng, Richard P.; Imperiali, Barbara

doi:10.1021/ja004292f

Cited by 61 publications

(63 citation statements)

References 32 publications

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“…2A) on the highly acid-labile TGT resin. The peptides were released from the resin with a C-terminal carboxylic acid and side-chain protection intact, and were subsequently derivatized to afford the corresponding C-terminal thioesters (Futaki et al 1997;Mezo et al 2001). The cpTyr building block was synthesized as previously described (Rothman et al 2003) to install a pTyr masked by the 1-(2-nitrophenyl)-ethyl (NPE) caging group.…”

Section: Resultsmentioning

confidence: 99%

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Vogel

Imperiali

2007

Protein Science

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Caged phosphopeptides and phosphoproteins are valuable tools for dissecting the dynamic role of phosphorylation in complex signaling networks with temporal and spatial control. Demonstrating the broad scope of phosphoamino acid caging for studying signaling events, we report here the semisynthesis of a photolabile precursor to the cellular migration protein paxillin, which is a complex, multidomain phosphoprotein. This semisynthetic construct provides a powerful probe for investigating the influence that phosphorylation of paxillin at a single site has on cellular migration. The 61-kDa paxillin construct was assembled using native chemical ligation to install a caged phosphotyrosine residue at position 31 of the 557-residue protein, and the probe includes all other binding and localization determinants in the paxillin macromolecule, which are essential for creating a native environment to investigate phosphorylation. Following semisynthesis, paxillin variants were characterized through detailed biochemical analyses and by quantitative uncaging studies.

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Section: Resultsmentioning

confidence: 99%

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Vogel

Imperiali

2007

Protein Science

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“…17 In the current studies we used the highly acid-labile carboxytrityl linker to the solid support (TGT-resin) to access the C-terminal thioester as previously introduced by our laboratory. 18 In a similar approach, other groups have employed 2-chlorotrityl linkers. 19 Highly acid-labile resin linkers allow orthogonal cleavage from the resin under mild acidic conditions without deprotection of amino acid side chains.…”

Section: Spps Of the Unglycosylated And Glycosylated Thioesters 1-28 mentioning

confidence: 99%

Semisynthesis of a Glycosylated Im7 Analogue for Protein Folding Studies

et al. 2005

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To establish a system to address questions concerning the influence of glycosylation on protein folding pathways, we have developed a semisynthetic route toward the immunity protein Im7. This four-helix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of Nterminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic Im7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.

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“…Because it has been reported that shortening the linker between domains favors domain-swapped oligomers (19, 20), we introduced a bias toward oligomerization in the BBA motif by varying the length of the 3-aa hinge region between secondary structural elements (21). Our approach for the discovery of homooligomers of peptides with ␤␤␣ supersecondary structure was based on a complementary design and screening process (21,22). BBAT1 (21, 22), a homooligomeric peptide, and the Gly9DAla analog, 1 (23) ( Table 1), share many characteristic features of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange.…”

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confidence: 99%

“…BBAT1 (21,22), a homooligomeric peptide, and the Gly9DAla analog, 1 (23) (Table 1), share many characteristic features of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange. Circular dichroism and 2D NMR experiments established that there are close contacts between residues in the helix and hairpin segments, that there are interhelical interactions, and that each monomer is in a symmetrical environment.…”

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X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture

Ali

Peisach

Allen

et al. 2004

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

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The x-ray crystal structure of an oligomeric miniprotein has been determined to a 1.2-Å resolution by means of multiwavelength anomalous diffraction phasing with selenomethionine analogs that retain the biophysical characteristics of the native peptide. Peptide 1, comprising ␣ and ␤ secondary structure elements with only 21 aa per monomer, associates as a discrete tetramer. The peptide adopts a previously uncharacterized quaternary structure in which ␣ and ␤ components interact to form a tightly packed and well defined hydrophobic core. The structure provides insight into the origins of the unusual thermal stability of the oligomer. The miniprotein shares many characteristics of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange, and possesses a buried surface area typical of native proteins. O ligomerization is a fundamental strategy for generating protein structural and functional complexity in nature. Many large proteins are believed to have arisen from oligomeric precursors that, by means of gene duplication and fusion, have become one encoded protein (1). Some examples are the ribosome anti-association factor eIF6 (2), the periplasmic binding proteins (3, 4), and the eightfold ␤͞␣ barrel (5). Functionally, many proteins (6) are inactive in the monomeric state but are active as a dimer or higher-order oligomer; examples include GCN4 (7) and MCP-1 (8).We are interested in studying the structural and functional advantages conferred by protein oligomerization. We thus set out to create miniproteins that would adopt a defined oligomeric state. Miniprotein models have been used to investigate biological processes and to model larger proteins through the incorporation of catalytic or other functionality (9-11). Oligomeric miniproteins serve as minimal model systems for quaternary structure formation. Moreover, they constitute platforms for probing whether more complex oligomeric structures might ultimately support function.The strategy for oligomerization was inspired by ''domain swapping,'' or the exchange of association partners, an important evolutionary mechanism for protein oligomerization (12)(13)(14)(15). This strategy has previously been used for the design of a coiled-coil domain-swapped dimer (16). The prototypic ␤␤␣ (BBA) motif BBA5 (Fig. 1) was chosen as the building block because it represents a discretely folded and structured miniprotein motif (17,18). This motif includes interactions between secondary structural elements, thus presenting an opportunity to favor similar interactions between monomers within an oligomer. Because it has been reported that shortening the linker between domains favors domain-swapped oligomers (19, 20), we introduced a bias toward oligomerization in the BBA motif by varying the length of the 3-aa hinge region between secondary structural elements (21). Our approach for the discovery of homooligomers of peptides with ␤␤␣ supersecondary structure was based on a complementary design and screening ...

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Oligomerization of Uniquely Folded Mini-Protein Motifs: Development of a Homotrimeric ββα Peptide

Cited by 61 publications

References 32 publications

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Semisynthesis of a Glycosylated Im7 Analogue for Protein Folding Studies

X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture

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