Engineering Enhanced Protein Stability through β‐Turn Optimization: Insights for the Design of Stable Peptide β‐Hairpin Systems

Simpson, Emma R.; Meldrum, Jill K.; Bofill, Roger; Crespo, Maria D.; Holmes, Elizabeth; Searle, Mark S.

doi:10.1002/ange.200500577

Cited by 12 publications

(29 citation statements)

References 68 publications

(27 reference statements)

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“…Accordingly, hairpins in folding nuclei show high Φ values in both the two strands and the turn between them. 18,[51][52][53] Our success in stabilizing the N2 domain of G3P supports previous studies 6,8,29,37,54,55 that suggested consensus sequence optimization of turns, possibly in combination with in vitro selection, as an excellent and simple means for increasing the stability of a protein.…”

Section: Discussionsupporting

confidence: 85%

“…These results are consistent with earlier substitutions in turns of α-spectrin SH3, 36 fibroblast growth factor-1, 37 lysozyme, 38 and ubiquitin. 6 …”

Section: Combination Of the Most Stable Designed Turn Sequencesmentioning

confidence: 97%

“…Resistant phage with stable guest protein are enriched in repeated cycles of in vitro proteolysis of the phage library, infection of Escherichia coli, and phage propagation. 6,39 This strategy was used here to stabilize G3P itself, in the absence of a guest protein. Comparisons of the B-factors for the crystal structures of wild-type G3P* (left) and 3T-G3P* (right).…”

Section: Optimization Of Turn 3 By In Vitro Selectionmentioning

confidence: 99%

“…The isolated N2 domain [residues 102-205 of the G3P of phage fd, extended by (His) 6 ] with the stabilizing mutation Q129H was used as the reference (pseudo-wild-type) protein for the rational design (N2′). All mutated variants of N2′ were expressed and purified as described previously.…”

Section: Expression and Purification Of Variants Of N2mentioning

confidence: 99%

See 3 more Smart Citations

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

Jakob

Zierer

Weininger

et al. 2010

Journal of Molecular Biology

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Section: Discussionsupporting

confidence: 85%

“…These results are consistent with earlier substitutions in turns of α-spectrin SH3, 36 fibroblast growth factor-1, 37 lysozyme, 38 and ubiquitin. 6 …”

Section: Combination Of the Most Stable Designed Turn Sequencesmentioning

confidence: 97%

Section: Optimization Of Turn 3 By In Vitro Selectionmentioning

confidence: 99%

Section: Expression and Purification Of Variants Of N2mentioning

confidence: 99%

See 2 more Smart Citations

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

Jakob

Zierer

Weininger

et al. 2010

Journal of Molecular Biology

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“…As one of the solutions to address the above mentioned problem, protein engineering has been successfully utilized to improve the thermostabilities of enzymes elsewhere (Gåseidnes et al, 2003;Korkegian et al, 2005;Simpson et al, 2005;Wunderlich et al, 2005;You et al, 2010). Unlike other stabilization methods (e.g.…”

Section: Q3mentioning

confidence: 99%

Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters

Huang

2015

Journal of Biotechnology

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Thermodynamic Analysis of β‐Sheet Secondary Structure by Backbone Thioester Exchange

et al. 2007

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Understanding the factors responsible for the stability of common protein secondary structures has been a longstanding goal. Analysis of b sheets has lagged behind the study of a helices because the development of suitable model systems has been more challenging for the former than for the latter.[1] Over the past decade, however, rules for the design of b sheets that fold autonomously in water have been elucidated, with a particular emphasis on two-stranded antiparallel b sheets ("b hairpins"), which represent a minimum increment of this secondary structure. [2,3] These model systems have proved useful for evaluating the contributions of factors such as side-chain-side-chain interactions, [4] interstrand linker composition, [5] strand length, [6] and strand number to the stability of b sheets. [7] As an outgrowth of this work, b hairpins have emerged as excellent platforms for the evaluation of noncovalent interactions that do not necessarily occur naturally in b sheets.[8] b Hairpins and hairpin-like molecules stabilized by cyclization are also attractive systems for biomedical applications and fundamental studies. [9] The use of designed peptides to probe the origins of bsheet stability, or to study noncovalent attractions between moieties that become spatially juxtaposed upon folding, requires the ability to determine the extent of b-sheet folding in solution. If only two conformational states are populated, unfolded and b sheet, then determining the population of these two states gives the folding equilibrium constant (K fold ), which provides insight into the stability of the folded state (DG fold = ÀR T lnK fold ). For proteins that adopt defined tertiary structures, conformational stability is often assessed by using heat or a chemical denaturant to disrupt the folded state while monitoring the extent of folding by a conformationally sensitive spectroscopic probe (for example, circular dichroism). This approach is convenient because globular proteins are typically completely folded near room temperature and in the absence of denaturant ("native conditions"), which establishes the spectroscopic signature of the folded state, and because it is often straightforward to identify the spectroscopic signature of a fully unfolded state generated at high temperature or high denaturant concentration.[10] In contrast, most of the autonomously folding b-sheet model systems described to date cannot be driven by changing the conditions to the limiting states. Therefore, identifying the spectroscopic signatures for the fully unfolded and fully folded states of b-sheet model systems has frequently required the preparation and characterization of distinct reference peptides or the implementation of elaborate data analysis techniques. [4][5][6][7][8] We have recently developed a new approach for studying the conformational stability of small proteins, [11] and here we describe the extension of this approach to b hairpins. This method involves polypeptide analogues in which one backbone amide group has been replaced b...

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Engineering Enhanced Protein Stability through β‐Turn Optimization: Insights for the Design of Stable Peptide β‐Hairpin Systems

Cited by 12 publications

References 68 publications

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters

Thermodynamic Analysis of β‐Sheet Secondary Structure by Backbone Thioester Exchange

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