Probing Backbone Hydrogen Bonds in Proteins by Amide‐to‐Ester Mutations

Sereikaitė, Vita; Jensen, Torben Pilegaard; Bartling, Christian R. O.; Jemth, Per; Pless, Stephan A.; Strømgaard, Kristian

doi:10.1002/cbic.201800350

Cited by 15 publications

(18 citation statements)

References 109 publications

(90 reference statements)

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“…Next, and in order to assess the contribution of the predicted backbone-mediated H-bond with ATP, we replaced the adjacent Val70 with the α-hydroxy analogue Vah using the nonsense suppression methodology (Nowak et al, 1998; England et al, 1999). Incorporating such a noncanonical amino acid replaces the backbone amide with a backbone ester bond, thus significantly reducing, but not eliminating, the H-bond acceptor propensity of the backbone carbonyl at the preceding position (Sereikaitė et al, 2018; Fig. 2 D).…”

Section: Resultsmentioning

confidence: 99%

“…Similarly, the conserved Thr184 had previously been suggested to contribute to ligand binding via both its side chain and its backbone carbonyl oxygen (Jiang et al, 2000; Roberts et al, 2008; Hattori and Gouaux, 2012; Kasuya et al, 2017). However, a functional verification of the notion of Lys69 and Thr184 contributing to ligand recognition via their backbone carbonyl oxygens had not been possible because site-directed mutagenesis does not allow backbone alterations (other than the introduction of Pro, which lacks a backbone NH moiety; Sereikaitė et al, 2018). Here, we thus turned to the nonsense suppression method to incorporate amide-to-ester mutations, constituting the first use of this approach in P2XRs.…”

Section: Discussionmentioning

confidence: 99%

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Molecular determinants for agonist recognition and discrimination in P2X2 receptors

Gasparri

Wengel

Grütter

et al. 2019

Journal of General Physiology

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Molecular determinants for agonist recognition and discrimination in P2X2 receptors

Gasparri

Wengel

Grütter

et al. 2019

Journal of General Physiology

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“…Apart from side chain contributions to ion channel function or pharmacology, those of the protein backbone can be probed through insertion of amide-to-ester modifications using aminoacylated tRNAs ( Fig. 4C; reviewed in Sereikaite et al 2018). This represents a key advantage over conventional site-directed mutagenesis, which cannot typically alter the H-bonding pattern of the protein backbone.…”

Section: Nonsense Suppression With Orthogonal Trnas In Xenopus Laevismentioning

confidence: 99%

The current chemical biology tool box for studying ion channels

Braun

Sheikh

Pless

2020

The Journal of Physiology

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Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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“…Non-canonical mutagenesis also provides many opportunities to alter the peptide backbone: changing its H-bonding, conformational, and secondary structure propensities. For example, amide to ester mutations have been employed to probe peptide-protein interactions (Eildal et al, 2013 ; Pedersen et al, 2014b ; Sereikaite et al, 2018 ). Mutation to ester replaces the amide H-bond donor with an acceptor and can therefore identify critical amide N-H interactions that drive folding and binding.…”

Section: Non-canonical One-at-a-time Mutagenesismentioning

confidence: 99%

Peptide Folding and Binding Probed by Systematic Non-canonical Mutagenesis

Rogers

2020

Front. Mol. Biosci.

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Many proteins and peptides fold upon binding another protein. Mutagenesis has proved an essential tool in the study of these multi-step molecular recognition processes. By comparing the biophysical behavior of carefully selected mutants, the concert of interactions and conformational changes that occur during folding and binding can be separated and assessed. Recently, this mutagenesis approach has been radically expanded by deep mutational scanning methods, which allow for many thousands of mutations to be examined in parallel. Furthermore, these high-throughput mutagenesis methods have been expanded to include mutations to non-canonical amino acids, returning peptide structure-activity relationships with unprecedented depth and detail. These developments are timely, as the insights they provide can guide the optimization of de novo cyclic peptides, a promising new modality for chemical probes and therapeutic agents.

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Probing Backbone Hydrogen Bonds in Proteins by Amide‐to‐Ester Mutations

Cited by 15 publications

References 109 publications

Molecular determinants for agonist recognition and discrimination in P2X2 receptors

Molecular determinants for agonist recognition and discrimination in P2X2 receptors

The current chemical biology tool box for studying ion channels

Peptide Folding and Binding Probed by Systematic Non-canonical Mutagenesis

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