Genetically Encoded Libraries of Constrained Peptides

Bosma, Tjibbe; Rink, Rick; Moosmeier, Markus A.; Moll, Gert N.

doi:10.1002/cbic.201900031

Cited by 20 publications

(22 citation statements)

References 48 publications

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“…1,2 Recently, it was shown that they can also be used in phage-and yeast-display techniques to select for high affinity binders of human cell surface receptors with medical relevance. [4][5][6] In addition, lanthipeptide libraries have been successfully used to screen for inhibitors of protein-protein interactions. 7 Lanthipeptide biosynthesis starts with the ribosomal synthesis of a genetically-encoded precursor peptide (LanA) that is matured by an enzymatic processing machinery.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic Studies of the Kinase Domains of Class IV Lanthipeptide Synthetases

et al. 2019

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Lanthipeptides, which belong to the superfamily of ribosomally synthesized and posttranslationally-modified peptides (RiPPs), are associated with interesting biological activities. Lanthipeptides can be subdivided into four classes that are defined by the characteristics of the corresponding posttranslational-modification enzymes. Class IV lanthipeptide synthetases consist of an N-terminal lyase, a central kinase, and a C-terminal cyclase domain. Here, we present the first in-depth characterization of such a kinase domain from the globisporin-maturation enzyme SgbL that originates from Streptomyces globisporus sp. NRRL B-2293. Catalytic residues were identified by alignments with homologs and structure modelling. Their roles were confirmed by employing proteins with Ala substitutions in in vitro modification and fluorescence polarization binding assays. Furthermore, the protein region that is binding the leader peptide was identified by hydrogen-deuterium exchange (HDX) -mass spectrometry experiments. By fusion of this protein region to the maltose binding protein, a protein was generated that can specifically bind the SgbA leader peptide, albeit with reduced binding affinity compared to full length SgbL. Combined, the results of this study provide a firmer grasp of how lanthipeptide biosynthesis is accomplished by class IV synthetases and suggest by homology analysis that biosynthetic mechanisms are similar in class III lanthipeptide processing enzymes.

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Section: Introductionmentioning

confidence: 99%

Mechanistic Studies of the Kinase Domains of Class IV Lanthipeptide Synthetases

et al. 2019

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“…It has been reported that rapid breakdown has limited the efficacy of many therapeutic peptides in complex biological fluids (de Vries et al, 2010). Cyclization can impose conformational constrains and then provide protection against proteolytic degradation as well as stabilize the conformation suitable for receptor binding (Bosma et al, 2019). Notably, a thioether link is more stable than a disulfide bond against hydrolytic decomposition, oxidation, or human serum (Tugyi et al, 2005; Rink et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Feasability of Introducing a Thioether Ring in Vasopressin by nisBTC Co-expression in Lactococcus lactis

Montalbán-López

Kuipers³

2019

Front. Microbiol.

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Introducing one or more intramolecular thioether bridges in a peptide provides a promising approach to create more stable molecules with improved pharmacodynamic properties and especially to protect peptides against proteolytic degradation. Lanthipeptides are compounds that naturally possess thioether bonds in their structure. The model lanthipeptide, nisin, is produced by Lactococcus lactis as a core peptide fused to a leader peptide. The modification machinery responsible for nisin production, including the Ser/Thr-dehydratase NisB and the cyclase NisC, can be applied for introducing a thioether bridge into peptides fused to the nisin leader peptide, e.g., to replace a disulfide bond. Vasopressin plays a key role in water homeostasis in the human body and helps to constrict blood vessels. There are two cysteine residues in the structure of wild type vasopressin, which form a disulfide bridge in the mature peptide. Here, we show it is possible to direct the biosynthesis of vasopressin variants in such a way that the disulfide bridge is replaced by a thioether bridge using the nisin modification machinery NisBTC, albeit at low efficiency. Vasopressin mutants were fused either to the nisin leader peptide directly (Type A), after the first three rings of nisin (Type B/C), or after full nisin (Type D). The type B strategy was optimal for expression. LC-MS/MS data verified the formation of a thioether bridge, which provides proof of principle for this modification in vasopressin. This is a first step prior to the necessary increase of the production yield and further purification of these peptides to finally test their biological activity in tissue and animal models.

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“…8b), the primary goal of this work was to demonstrate how NGS and coexpression of fluorescent proteins can be used to enhance data analysis of display libraries in general, rather than to provide an optimized peptide sequence for binding PLA. To further optimize affinity and specificity of peptides for PLA, it could be useful to employ a constrained peptide library, as they have been shown to improve both affinity and specificity [19]. Specificity for PLA, for instance over other plastics, can also be improved by incorporation of negative sorting steps in future studies.…”

Section: Discussionmentioning

confidence: 99%

“…The increase in cysteine after sorting is particularly interesting since it could have both positive and negative effects on the peptide library. Cysteine could help constrain any structural component of the peptide that aids in binding affinity or specificity by creating disulfide bonds that increase rigidity [19]. However, when several cysteine residues are present in the library, unwanted intermolecular disulfide bonding could also occur, which could cause clumping of the displayed cells and interfere with isolation of individual peptides within the population for characterization.…”

Section: Analysis Of Library Diversitymentioning

confidence: 99%

The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries

et al. 2019

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Background: Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. Results: In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. Conclusions: We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.

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Genetically Encoded Libraries of Constrained Peptides

Cited by 20 publications

References 48 publications

Mechanistic Studies of the Kinase Domains of Class IV Lanthipeptide Synthetases

Mechanistic Studies of the Kinase Domains of Class IV Lanthipeptide Synthetases

Feasability of Introducing a Thioether Ring in Vasopressin by nisBTC Co-expression in Lactococcus lactis

The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries

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