Minimization and Optimization of Designed β-Hairpin Folds

Andersen, Niels H.; Olsen, Katherine A.; Fesinmeyer, R. Matthew; Tan, Xu; Hudson, F. Michael; Eidenschink, Lisa A.; Farazi, S.

doi:10.1021/ja054971w

Cited by 117 publications

(187 citation statements)

References 40 publications

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“…All peptides of the form "(acyl)-W-loop-WTG…" (where "loop" is any sequence capable of forming a tight turn; e.g., IpGL, IHGK, ENGR, etc.) exhibited the remarkable stability and diagnostic spectroscopic features expected of stable hairpin folds with cross-strand Trp/Trp pairs at a loop-flanking non-H-bonded position, including a strong exciton couplet visible by circular dichroism (CD) (λ max ¼ 228 nm) (10,11,13,23). However, unlike prior examples (7, 10-13, 22, 23, 29, 30) of turn-flanking Trp/ Trp interactions (SI Appendix), in which the upfield chemical shifts observed for the Hε3 and Hβ3 protons for the "edge" tryptophan were observed for the N-terminal Trp, in the acyl-W-loop-WTG species, the C-terminal Trp was the edge species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The NOESY spectra for NMR ensemble generation of Ac-WITVTIHGKKIRVWTG-NH 2 was acquired on a 750 MHz NMR at 280 K with a mixing time of 120 ms. NOE intensities were converted to distant ranges using an in-house program (di8) which corrects for multiple chemically equivalent protons and sharp aromatic peaks (42,43). NMR structure generation and acceptance criteria were described previously (13) No structures with NOE constraint violations greater than 0.35 Å were included in the accepted ensembles. Ensemble statistics and the lists of NOE constraints appear in the SI Appendix.…”

Section: Methodsmentioning

confidence: 99%

“…The key discoveries that improved β-hairpin stabilities outside of protein contexts have been sequences with good turn propensities, for example D-Pro-Gly (pG) (6), heterochiral pP (7), and Aib-Gly (8) [or less favorably, Asn-Gly (NG) (5,9)] and the incorporation of optimized cross-strand pairings [most notably Trp/Trp pairs flanking the turn (10)(11)(12)(13)(14)]. However, longer hairpin models are typically still frayed at the termini; to date, fully folded spectroscopic reference values have only been attained via cyclization (15)(16)(17).…”

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confidence: 99%

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Stabilizing capping motif for β-hairpins and sheets

Kier

Shu²,

Eidenschink³

et al. 2010

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

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158

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Although much has been learned about the design of models of β-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most β-hairpin peptides. In the case of hairpin capping, nature did not provide guidance for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally applicable, "unnatural" beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a net contribution of 6 þ kJ∕mol to β-hairpin stability, surpassing all other interactions that stabilize β-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel β-strands and reduces fraying at the ends of hairpins and other β-sheet models. Utilizing this motif, 10-to 22-residue peptide scaffolds of defined stereochemistry that are greater than 98% folded in water have been prepared. The β-cap can also be used to staple together short antiparallel β-strands connected by a long flexible loop.beta sheets | capping stabilization | peptide hairpins | Trp/Trp interactions C apping motifs are well-known features of protein and peptide secondary structure; specifically, terminal alpha helix caps (especially N caps) are common both in proteins and designed peptides (1-4). The basis for helix capping is straightforward: countering the helix macrodipole and providing additional H-bonding interactions (1). Caps increase the fold population of isolated α-helical peptides and have been a boon to the design of α-helix models. β-Structures have capping requirements wholly different from those of helices; the ends of canonical β-sheets and hairpins do not have dipole moments or unsatisfied H bonds.The a priori design of model β-sheet systems (5), focused on hairpins, has lagged behind that of α-helix counterparts. The key discoveries that improved β-hairpin stabilities outside of protein contexts have been sequences with good turn propensities, for example D-Pro-Gly (pG) (6), heterochiral pP (7), and Aib-Gly (8) [or less favorably, Asn-Gly (NG) (5, 9)] and the incorporation of optimized cross-strand pairings [most notably Trp/Trp pairs flanking the turn (10-14)]. However, longer hairpin models are typically still frayed at the termini; to date, fully folded spectroscopic reference values have only been attained via cyclization (15-17). With the exception of cyclization, mutations at terminal sites have yielded only modest changes in hairpin stability; terminal coulombic effects (ΔΔG U ¼ 1.5-2.5 kJ∕mol) standing as the only generally observed capping effect (11,18,19). Pi-cation interactions have also been shown to provide significant hairpin stabilization (20), but instances in which the interaction appears near the ends of hairpins have provided only marginal stability increases (18,(21)(22)(23)). An unnatural π-cation interaction has also been shown to stabilize the turn in a tripeptide (24). There has been limited evidence for hairpin fold sta...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Stabilizing capping motif for β-hairpins and sheets

Kier

Shu²,

Eidenschink³

et al. 2010

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

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“…Trpzips are a class of highly folded b-hairpins that are defined by the presence of cross-strand diagonally oriented Trp/Trp pairs on one face of the hairpin. [1,5,10] The minimal and highly stable trpzip structure has emerged as a preferred model system for computational and experimental studies of protein folding. [11][12][13] We have proposed that trpzip-type peptides (or tandem fusions of such peptides) could serve as a minimal protein scaffold for molecular recognition in the cytoplasm of live cells.…”

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confidence: 99%

“…[15] The template for our initial "extended trpzip" library was a 20-mer version of the highly folded 16-mer trpzip HP5W4. [10] The 20-mer contained two additional pairs of residues, one random and one threonine, genetically inserted after what would otherwise have been the second and 14th residues of HP5W4. ; here X represents all 20 amino acids, and the residues inserted relative to HP5W4 are underlined.…”

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In Vivo Screening Identifies a Highly Folded β‐Hairpin Peptide with a Structured Extension

2007

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The conventional approach for developing folded peptides involves chemical synthesis of systematically modified peptide variants and individual characterization by circular dichroism and NMR spectroscopy. [1][2][3][4][5] The use of synthetic peptides and reliance on low-throughput characterization techniques necessarily restricts the sequence diversity that can be explored, though efforts have been made to overcome this limitation. [6][7][8] We have recently described an approach that enables us to asses the ability of peptides to fold into b-hairpins in the cytoplasm of live cells.[9] Our strategy entails the recombinant expression of a peptide gene fused in frame between flanking genes encoding a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). If a particular peptide sequence adopts a folded structure, CFP and YFP are brought into closer proximity and exhibit a higher efficiency of fluorescence resonance energy transfer (FRET). Higher FRET efficiency enhances the YFP (acceptor) fluorescence at the expense of the CFP (donor) fluorescence. Imaging of plates harboring colonies of transformed bacteria provides the YFP and CFP fluorescence emission intensities for each colony, and thus peptides that are highly folded in vivo can be distinguished from those that are not.As will be described in this manuscript, this FRET-based approach also provides a versatile method for screening large libraries of peptide sequences for highly structured variants. We have applied this strategy to the development of a version of a "tryptophan zipper" (trpzip)-type b-hairpin [1] with a structured extension. Trpzips are a class of highly folded b-hairpins that are defined by the presence of cross-strand diagonally oriented Trp/Trp pairs on one face of the hairpin. [1,5,10] The minimal and highly stable trpzip structure has emerged as a preferred model system for computational and experimental studies of protein folding. [11][12][13] We have proposed that trpzip-type peptides (or tandem fusions of such peptides) could serve as a minimal protein scaffold for molecular recognition in the cytoplasm of live cells. [9, 14] With this goal in mind, we sought to employ our screening strategy to identify a candidate trpziptype peptide with high fold stability in vivo. A similar approach has previously been used to increase the thermal stability of engineered immunoglobulin V L domains. [15] The template for our initial "extended trpzip" library was a 20-mer version of the highly folded 16-mer trpzip HP5W4.[10]The 20-mer contained two additional pairs of residues, one random and one threonine, genetically inserted after what would otherwise have been the second and 14th residues of HP5W4. ; here X represents all 20 amino acids, and the residues inserted relative to HP5W4 are underlined. Assuming b-strand conformation, the randomized positions would be directed towards the face of HP5W4 that harbors the interdigitated Trp side chains.Escherichia coli was transformed with the gene library, and 6 10 3 colonies on 10 Petri dis...

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Hairpin structure stability plays a role in the activity of two antimicrobial peptides

et al. 2016

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Many naturally occurring antimicrobial peptides are amphipathic with a β-hairpin conformation stabilized by cross-strand disulfides across the associated β-strands. Here, we show that the disulfides are not essential. Other structuring means such as better β-turns and non-covalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity. Our results also demonstrate that the hairpin turn region may play a role in membrane recognition for at least one member of this class, since a homodimeric turnless β-sheet analog showed no antimicrobial activity. We also examined the effects of N-terminal fatty acid adducts on antimicrobial peptides. Surprisingly, the large hydrophobic carboxylic moieties examined completely eliminated the antimicrobial activity of previously active β-hairpin peptides.

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Minimization and Optimization of Designed β-Hairpin Folds

Cited by 117 publications

References 40 publications

Stabilizing capping motif for β-hairpins and sheets

Stabilizing capping motif for β-hairpins and sheets

In Vivo Screening Identifies a Highly Folded β‐Hairpin Peptide with a Structured Extension

Hairpin structure stability plays a role in the activity of two antimicrobial peptides

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