Photo-control of peptide helix content by an azobenzene cross-linker: steric interactions with underlying residues are not critical

Kumita, Janet R.; Flint, Daniel G.; Smart, Oliver S.; Woolley, G. Andrew

doi:10.1093/protein/15.7.561

Cited by 37 publications

(36 citation statements)

References 51 publications

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“…Previous studies with azobenzene derivatives have shown that cross-linking two residues within an α-helix leads to a light-induced change in helical content (3,13,16,18,20,25). Following this strategy, we introduced cross-linking sites in three α-helices, two of them located in the DNA binding subdomain (C89-C96 and C129-C133, respectively) and one of them in the catalytic subdomain (C147-C151) ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown that peptides and proteins can be modified with such bifunctional azobenzene derivatives and that these chemically modified peptides and proteins can be induced to change their conformation and/or their activity in a reversible manner by illumination with light (16,(18)(19)(20)(21)(22)(23)(24)(25). To date, this is the most promising generally applicable method to produce photoswitchable proteins, in particular when structural information is available (26).…”

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confidence: 99%

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Controlling the enzymatic activity of a restriction enzyme by light

Schierling

Noël

Wende

et al. 2009

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

167

164

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For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis-or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when crosslinked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, >30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V max rather than K m .azobenzene | DNA cleavage | endonuclease | photoswitch | PvuII P roteins exist in nature whose activity can be controlled by light; perhaps one of the best known examples is rhodopsin, which is regulated by the cis∕trans isomerization of its cofactor retinal. For many biological applications it would be desirable to selectively switch the activity of a protein on and off by light in a similar manner (1). This could be accomplished by the introduction of a photosensitive compound into the protein of interest. Recent developments in photosensitive compounds such as the azobenzene derivatives have made the scenario a reality. Azobenzene can be reversibly isomerized between the extended trans-and the more compact cis-configuration by illumination with UV (trans → cis) or blue-light (cis → trans) as well as by thermal relaxation (cis → trans) (2-4). Four generally applicable approaches have been used to introduce azobenzene groups into peptides or proteins: (i) incorporation during peptide synthesis (5-8), (ii) incorporation during in vitro translation (9, 10), (iii) incorporation in vivo by using an orthogonal tRNA/aminoacyl tRNA synthetase pair specific for phenylalanine-4′-azobenzene (11), and (iv) chemical modification of peptides and proteins (3,12,13). Another more specific approach is to use azobenzenemodified ligands (e.g., inhibitors) for proteins (14,15). Chemical modification, the most widely used of these approaches, can be done with mono-or bifunctional azobenzene derivatives. Modification with monofunctional azobenzene derivatives relies on steric effects (e.g., interference with ligand binding), whereas modification with bifunctional azobenzene derivat...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Controlling the enzymatic activity of a restriction enzyme by light

Schierling

Noël

Wende

et al. 2009

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

167

164

View full text Add to dashboard Cite

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“…The panel examined several crosslinking positions in the peptide sequence (avoiding the interacting surface of the peptide), different crosslinking distances, and the introduction of the nonproteinogenic amino acids a-aminoisobutyric acid (Aib), and 1-and 2-naphtylalanine (1-and 2-Nal). These elements are useful in design of a-helix mimetics (Becerril and Hamilton, 2007;Edwards and Wilson, 2011), stapled cell-penetrating peptides (Moellering et al, 2009;Verdine and Hilinski, 2012;Walensky et al, 2004), structurally photoswitchable a-helical peptides (Kumita et al, 2000(Kumita et al, , 2002Woolley, 2005), and photo-triggered peptide inhibitors (Kneissl et al, 2008). Several peptides were N-capped with a succinyl group to stabilize the structure with the aim of influencing the uptake by cells.…”

Section: Design Synthesis and Photoswitching Of Tlsmentioning

confidence: 99%

“…The imposition of a helical turn, especially near the center of a peptide sequence, can help nucleate the formation of the helical structure. It has been reasoned that the introduction of bulky Val and Aib beneath the azobenzene crosslinker might increase the conformational photoswitching via a steric clash with transazobenzene (Kumita et al, 2000(Kumita et al, , 2002. We included the Val-Aib motif in several peptides of our collection to assess its effects on the performance of photoswitchable peptide inhibitors (i.e., on interaction and not only on structure).…”

Section: Chemistry and Biologymentioning

confidence: 99%

Absence of a Stable Secondary Structure Is Not a Limitation for Photoswitchable Inhibitors of β-Arrestin/β-Adaptin 2 Protein-Protein Interaction

Martín‐Quirós¹,

Nevola²,

Eckelt³

et al. 2015

Chemistry & Biology

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Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a β-arrestin P-long 20-mer peptide (BAP-long) sequence to assess the relevance of secondary structure in their interaction with β-adaptin 2 and to identify the design requirements for photoswitchable inhibitors of PPI (PIPPIs). We observe that flexible structures show a greater inhibitory capacity and enhanced photoswitching ability and that the absence of helical structures in free inhibitor peptide is not a limitation for PIPPI candidates. Therefore, our PIPPIs expand the field of potential inhibitors of PPIs to the wide group of flexible peptides, and we argue against using a stable secondary structure as a sole criterion when designing PIPPI candidates.

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“…The extent to which this is achieved depends critically on where the switch is placed and how well isomerization is coupled to protein conformational change. Incorrect placement can lead to inconsequential changes in conformation or to switches becoming locked in the cis or trans state [15][16][17] .…”

Section: Introductionmentioning

confidence: 99%