2019
DOI: 10.1021/acs.biochem.9b00067
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Stabilizing and Understanding a Miniprotein by Rational Redesign

Abstract: Miniproteins reduce the complexity of the protein-folding problem allowing systematic studies of contributions to protein folding and stabilization. Here, we describe the rational redesign of a miniprotein, PP, comprising a polyproline-II helix, loop and  helix. The redesign provides a de novo framework to interrogate non-covalent interactions. Optimized PP has significantly improved thermal stability with a midpoint unfolding temperature (TM) of 51C. Its NMR structure indicates a higher density of stabili… Show more

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Cited by 4 publications
(5 citation statements)
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“…As an initial evaluation of the impacts of backbone alteration on tertiary folded structure, we subjected each sequence to circular dichroism (CD) spectroscopy. CD scans at 20 °C performed on samples consisting of 100 μM peptide in pH 7.4 phosphate buffered saline (PBS) showed minima at ∼208 and ∼222 nm across the series (Figure 2A), consistent with prior reported results for the parent peptide 1 [12b] . This suggests the overall tertiary fold of the domain is intact across the variants, a hypothesis corroborated further by NMR analysis detailed below.…”
Section: Resultssupporting
confidence: 88%
See 1 more Smart Citation
“…As an initial evaluation of the impacts of backbone alteration on tertiary folded structure, we subjected each sequence to circular dichroism (CD) spectroscopy. CD scans at 20 °C performed on samples consisting of 100 μM peptide in pH 7.4 phosphate buffered saline (PBS) showed minima at ∼208 and ∼222 nm across the series (Figure 2A), consistent with prior reported results for the parent peptide 1 [12b] . This suggests the overall tertiary fold of the domain is intact across the variants, a hypothesis corroborated further by NMR analysis detailed below.…”
Section: Resultssupporting
confidence: 88%
“…Collectively, the statistics obtained for peptides 1 – 5 show that conformational preferences inherent to the various artificial turn inducers in the context of hairpins hold in the non‐hairpin loop of the oPPα domain (Figure 4). The β‐turn in the NMR ensemble for prototype peptide 1 is predominantly type I, consistent with the both the prior reported NMR structure [12b] as well as a crystal structure of a homologous loop sequence in the context of avian pancreatic polypeptide, [17] a closely related fold. The Aib‐Gly motif in peptide 2 leads to populations of both mirror image (type I′/II′) and non‐mirror image (type I) turns, while the d Pro‐Gly moiety in peptide 3 predisposes the loop toward mirror image turns.…”
Section: Resultssupporting
confidence: 88%
“…Moreover, the relatively extensive structure of miniproteins allows the creation of molecules with advanced functions. Miniprotein folding can be driven by the formation of a hydrophobic core, metal binding, or cysteine disulfides (Huang et al, 2016; Porter Goff et al, 2019; Zhu et al, 2003). Miniproteins occur in nature, can be engineered domains of native proteins, or are available as created artificially using an in silico approach (Gates et al, 2018; Korendovych and DeGrado, 2020; Rocklin et al, 2017).…”
Section: Introductionmentioning
confidence: 99%
“…Therefore, the rational design and engineering of proteins have gained a lot of attention. In particular, miniproteins, i.e., proteins with molecular mass not exceeding 10 kDa exhibiting a well-defined three-dimensional structure, have recently become an emerging class of molecules. They combine several features that make them valuable and attractive compounds. First, modern computer-aided techniques allow efficient design of new structures. Second, miniproteins can be obtained using a solid-phase peptide synthesis approach or overexpressed in cells.…”
mentioning
confidence: 99%
“…In particular, miniproteins, i.e., proteins with molecular mass not exceeding 10 kDa exhibiting a well-defined three-dimensional structure, have recently become an emerging class of molecules. 1 5 They combine several features that make them valuable and attractive compounds. First, modern computer-aided techniques allow efficient design of new structures.…”
mentioning
confidence: 99%