High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation

He, Lijuan; Hoffmann, Andrew R.; Serrano, Christopher M.; Hristova, Kalina; Wimley, William C.

doi:10.1016/j.jmb.2011.07.004

Cited by 24 publications

(23 citation statements)

References 51 publications

(63 reference statements)

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“…1, which contains 16,200 members, was synthesized as one bead–one sequence library, as described in detail elsewhere 24–27,45 . To determine the P:L ratio to be used in the high–throughput screen, we tested a sample of the iterative library and compared it to the original library at a low stringency of P:L = 1:200.…”

Section: Resultsmentioning

confidence: 99%

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Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

Krauson

Wimley

et al. 2013

ACS Chem. Biol.

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We previously reported the de novo design of a combinatorial peptide library that was subjected to high–throughput screening to identify membrane permeabilizing antimicrobial peptides that have β–sheet–like secondary structure. Those peptides do not form discreet pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24–26–residue, 16,200 member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non–helical secondary structure. We used a new high–throughput assay to identify members that self–assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore–formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore–forming peptides known. Furthermore, they are not α–helical, which makes them unusual, as most of the highly potent pore–forming peptides are amphipathic α–helices. Here we demonstrate that this synthetic molecular evolution–based approach, taken together with the new high–throughput tools we have developed, enables the identification, refinement and optimization of unique membrane active peptides.

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Section: Resultsmentioning

confidence: 99%

“…The iterative combinatorial peptide library was synthesized on Tentagel NH 2 macrobeads (300 mm diameter) as described in detail elsewhere 24,26,27,45 . Validation of library synthesis was done using mass spectrometry, HPLC and Edman sequencing.…”

Section: Methodsmentioning

confidence: 99%

Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

Krauson

Wimley

et al. 2013

ACS Chem. Biol.

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“…In a recent study that addresses the specificity of dimerization motifs (108), we used an SDS-PAGE based high throughput screen to select strongly homo-dimerizing sequences from a combinatorial library based on the rat neu (ErbB2) TM domain. In the 3,888-member peptide library there were a very large number of recognizable dimerization motifs.…”

Section: Dimerization Motifsmentioning

confidence: 99%

Transmembrane helix dimerization: Beyond the search for sequence motifs

Wimley

Hristova

2012

Biochimica et Biophysica Acta (BBA) - Biomembranes

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148

158

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Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex.

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“…A recent NMR study shows that the membrane-spanning helices of ErbB4 form a parallel dimer in lipid bicelles and undergo a structural adjustment to form a network of intermonomeric polar contacts and provide entropic enhancement for the weak helix–helix interactions (Bocharov et al 2012). Interestingly, when thousands of peptides, based on the TMD of ErbB2 (Neu) were screened for their dimerization properties, some of the sequences were found to activate the ErbB2 kinase significantly more than the wild-type sequence (He et al 2011), further highlighting the potential importance of this region. In addition, the isolated FGFR TMD was shown to dimerize in the absence of the extracellular domain or ligands (Li et al 2005).…”

Section: Eph Receptors and Ephrinsmentioning

confidence: 99%

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

Barton

Dalton

Seegar

et al. 2014

Cold Spring Harbor Perspectives in Biology

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The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.

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High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation

Cited by 24 publications

References 51 publications

Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

Transmembrane helix dimerization: Beyond the search for sequence motifs

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

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