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Computational design of highly signalling-active membrane receptors through solvent-mediated allosteric networks

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Chen, K.-Y. M., Lai, J. K., Rudden, L. S. P., Wang, J., Russell, A. M., Conners, K., Rutter, M. E., Condon, B., Tung, F., Kodandapani, L., Chau, B., Zhao, X., Benach, J., Baker, K., Hembre, E. J., Barth, P.·2025·Nature Chemistry
DOI 10.1038/s41557-024-01719-2PMC PMC11882447

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Abstract

Protein catalysis and allostery require the atomic-level orchestration and motion of residues and ligand, solvent and protein effector molecules. However, the ability to design protein activity through precise protein–solvent cooperative interactions has not yet been demonstrated. Here we report the design of 14 membrane receptors that catalyse G protein nucleotide exchange through diverse engineered allosteric pathways mediated by cooperative networks of intraprotein, protein–ligand and –solvent molecule interactions. Consistent with predictions, the designed protein activities correlated well with the level of plasticity of the networks at flexible transmembrane helical interfaces. Several designs displayed considerably enhanced thermostability and activity compared with related natural receptors. The most stable and active variant crystallized in an unforeseen signalling-active conformation, in excellent agreement with the design models. The allosteric network topologies of the best designs bear limited similarity to those of natural receptors and reveal an allosteric interaction space larger than previously inferred from natural proteins. The approach should prove useful for engineering proteins with novel complex protein binding, catalytic and signalling activities.

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Figures

Fig. 1 Rational design of signalling activity through de novo solvent-mediated interaction networks. a, Dynamic remodelling of the solvent-mediated allosteric network during the activation of the μ-opioid receptor (mOR). The positions of water molecules and hydrogen bonds were extracted from the inactive (Protein Data Bank (PDB): 4DKL) and active (PDB: 5C1M) mOR structures. b, Schematic representation of a water-mediated hydrogen bond network bridging four helices designed using the SPaDES software. De novo amino acid sequences, conformations and water positions were searched concurrently for optimal water-mediated hydrogen bond connectivity at the helical interface. c, Snapshot of a low-energy water-mediated hydrogen bond network ensemble in the core of a GPCR structure. d–f, Computational design strategies for enhancing protein activity and conformational stability following three criteria: shifts in conformational energies (d), increased water-mediated dynamic contacts in the active state (e) or weakened ion-mediated locks in the inactive state (f).
Fig. 1 Rational design of signalling activity through de novo solvent-mediated interaction networks. a, Dynamic remodelling of the solvent-mediated allosteric network during the activation of the μ-opioid receptor (mOR). The positions of water molecules and hydrogen bonds were extracted from the inactive (Protein Data Bank (PDB): 4DKL) and active (PDB: 5C1M) mOR structures. b, Schematic representation of a water-mediated hydrogen bond network bridging four helices designed using the SPaDES software. De novo amino acid sequences, conformations and water positions were searched concurrently for optimal water-mediated hydrogen bond connectivity at the helical interface. c, Snapshot of a low-energy water-mediated hydrogen bond network ensemble in the core of a GPCR structure. d–f, Computational design strategies for enhancing protein activity and conformational stability following three criteria: shifts in conformational energies (d), increased water-mediated dynamic contacts in the active state (e) or weakened ion-mediated locks in the inactive state (f).CC BY · PubMed Central
Fig. 2 Computational-designed GPCRs with a wide range of water-mediated interaction networks at switchable TMH interfaces. a, Backbone representation of a GPCR scaffold highlighting static (dark grey) and switchable (light grey) TM helices. b, Relationship between the total number of buried water molecules mediating polar contacts and those at static–switchable TMH interfaces. Data for the native A2AR (light grey) are included for comparison. Designs with a higher and lower number of water-mediated static–switchable hydrogen bonds than A2AR are classified as Hyd_high and Hyd_low, respectively. c,d, Hydrogen bond network in the TMH core regions of the Hyd_low2 (c) and Hyd_high4 (d) structures. The positions of the residues highlighted as stick models are indicated by Ballesteros–Weinstein notation. e, Adenosine-induced activity of the designed receptor variants (as a percentage of adenosine-induced WT A2AR activity) as a function of the number of solvent-mediated interactions at static–switchable TMH interfaces. A baseline for the native A2AR (light grey) is included for comparison. The data are presented as the mean ± standard error of the mean (s.e.m.) for n = 3 independent experiments. Source data
Fig. 2 Computational-designed GPCRs with a wide range of water-mediated interaction networks at switchable TMH interfaces. a, Backbone representation of a GPCR scaffold highlighting static (dark grey) and switchable (light grey) TM helices. b, Relationship between the total number of buried water molecules mediating polar contacts and those at static–switchable TMH interfaces. Data for the native A2AR (light grey) are included for comparison. Designs with a higher and lower number of water-mediated static–switchable hydrogen bonds than A2AR are classified as Hyd_high and Hyd_low, respectively. c,d, Hydrogen bond network in the TMH core regions of the Hyd_low2 (c) and Hyd_high4 (d) structures. The positions of the residues highlighted as stick models are indicated by Ballesteros–Weinstein notation. e, Adenosine-induced activity of the designed receptor variants (as a percentage of adenosine-induced WT A2AR activity) as a function of the number of solvent-mediated interactions at static–switchable TMH interfaces. A baseline for the native A2AR (light grey) is included for comparison. The data are presented as the mean ± standard error of the mean (s.e.m.) for n = 3 independent experiments. Source dataCC BY · PubMed Central
Fig. 3 Designed receptor stability and function strongly depend on the density of solvent-mediated interactions at switchable TMH interfaces. a, Constitutive activity (as a percentage of maximal agonist-induced WT A2AR activity) as a function of the calculated conformational energy difference between the active and inactive states relative to A2AR. The reported energy difference was calculated from the average of the ten lowest-energy inactive- and active-state structures (Supplementary Table 3). The best linear fit to the data is shown as a dotted line (correlation coefficient r = 0.91). b, Measured constitutive activity of the designed receptor variants (as a percentage of adenosine-induced A2AR activity) as a function of the number of solvent-mediated interactions at static–switchable TMH interfaces. c,d, Apparent stability of Hyd_low (c) and Hyd_high (d) receptors compared with A2AR, measured as the functional half-life of adenosine-bound receptor variants incubated at 37 °C. The values for all of the designs are given in Supplementary Table 4. In b–d, the data are presented as the mean ± s.e.m. for n = 3 independent experiments. Source data
Fig. 3 Designed receptor stability and function strongly depend on the density of solvent-mediated interactions at switchable TMH interfaces. a, Constitutive activity (as a percentage of maximal agonist-induced WT A2AR activity) as a function of the calculated conformational energy difference between the active and inactive states relative to A2AR. The reported energy difference was calculated from the average of the ten lowest-energy inactive- and active-state structures (Supplementary Table 3). The best linear fit to the data is shown as a dotted line (correlation coefficient r = 0.91). b, Measured constitutive activity of the designed receptor variants (as a percentage of adenosine-induced A2AR activity) as a function of the number of solvent-mediated interactions at static–switchable TMH interfaces. c,d, Apparent stability of Hyd_low (c) and Hyd_high (d) receptors compared with A2AR, measured as the functional half-life of adenosine-bound receptor variants incubated at 37 °C. The values for all of the designs are given in Supplementary Table 4. In b–d, the data are presented as the mean ± s.e.m. for n = 3 independent experiments. Source dataCC BY · PubMed Central
Fig. 4 Polar interaction networks connecting static and switchable TMHs in the Hyd_low1 design and A2AR. a, Cartoon representation of the Hyd_low1 receptor active-state model, where the adenosine ligand and side chains in the vicinity of the designed mutations are highlighted as stick models. b,c, Enlarged views of the designed region in Hyd_low1 (b) and A2AR (c). Polar contacts are represented by red dotted lines. Residues are numbered using the Ballesteros–Weinstein notation.
Fig. 4 Polar interaction networks connecting static and switchable TMHs in the Hyd_low1 design and A2AR. a, Cartoon representation of the Hyd_low1 receptor active-state model, where the adenosine ligand and side chains in the vicinity of the designed mutations are highlighted as stick models. b,c, Enlarged views of the designed region in Hyd_low1 (b) and A2AR (c). Polar contacts are represented by red dotted lines. Residues are numbered using the Ballesteros–Weinstein notation.CC BY · PubMed Central
Fig. 5 The designed Hyd_high7 structure adopts an unforeseen activated conformation. a, Backbone representation of the designed Hyd_high7 structure bound to the ligand agonist CGS21680 and N-terminally fused to a T4Lcp variant. b, Cytoplasmic view of the following superimposed structures: antagonist-bound A2AR (PDB: 3EML, yellow), agonist-bound, computationally designed Hyd_high7 (green), Gt peptide-bound opsin active state (PDB: 3DQB, blue), and agonist- and mini-Gs-bound A2AR thermostabilized by alanine scanning (PDB: 5G53, magenta). The structures are ranked according to the magnitude of the conformational changes towards the fully active state (back arrow). c–e, Conformations of key conserved microswitches involved in receptor activation (in addition to the reference structures described in panel b, the following structure is also represented: agonist-bound A2AR thermostabilized by alanine scanning (PDB: 2YDO, grey)): the TMH3–TMH5–TMH6 interface around the W6.48 toggle switch in the receptor TM core (c), the TM3–TMH5–TMH6 interface around the intracellular DR3.50Y motif (d) and the TMH6–TMH7 interface around the intracellular N7.49PXXY7.53 motif (e). Conformational changes between inactive, partially active and fully active states are indicated by distinct black arrows. f–k, Comparison of the conformation of the designed (black) and neighbouring native (grey) residue microswitches in the agonist-bound designed Hyd_high7 structure (f) and the agonist- and Gs-bound Hyd_high7 design model (g) as well as the TMH5–TMH6 core interface (h), the TMH6–TMH7 intracellular interface with bound Gs (magenta) (i), the TMH1–TMH2–TMH3 core interface at the Na+ ion-conserved binding site (j) and the TMH3–TMH6–TMH7 intracellular interface (k).
Fig. 5 The designed Hyd_high7 structure adopts an unforeseen activated conformation. a, Backbone representation of the designed Hyd_high7 structure bound to the ligand agonist CGS21680 and N-terminally fused to a T4Lcp variant. b, Cytoplasmic view of the following superimposed structures: antagonist-bound A2AR (PDB: 3EML, yellow), agonist-bound, computationally designed Hyd_high7 (green), Gt peptide-bound opsin active state (PDB: 3DQB, blue), and agonist- and mini-Gs-bound A2AR thermostabilized by alanine scanning (PDB: 5G53, magenta). The structures are ranked according to the magnitude of the conformational changes towards the fully active state (back arrow). c–e, Conformations of key conserved microswitches involved in receptor activation (in addition to the reference structures described in panel b, the following structure is also represented: agonist-bound A2AR thermostabilized by alanine scanning (PDB: 2YDO, grey)): the TMH3–TMH5–TMH6 interface around the W6.48 toggle switch in the receptor TM core (c), the TM3–TMH5–TMH6 interface around the intracellular DR3.50Y motif (d) and the TMH6–TMH7 interface around the intracellular N7.49PXXY7.53 motif (e). Conformational changes between inactive, partially active and fully active states are indicated by distinct black arrows. f–k, Comparison of the conformation of the designed (black) and neighbouring native (grey) residue microswitches in the agonist-bound designed Hyd_high7 structure (f) and the agonist- and Gs-bound Hyd_high7 design model (g) as well as the TMH5–TMH6 core interface (h), the TMH6–TMH7 intracellular interface with bound Gs (magenta) (i), the TMH1–TMH2–TMH3 core interface at the Na+ ion-conserved binding site (j) and the TMH3–TMH6–TMH7 intracellular interface (k).CC BY · PubMed Central
Fig. 6 Hyd-high7 is built of a synthetic allosteric interaction network distant from those of natural GPCRs. a,b, Solvent-mediated polar interaction networks at static–switchable TMH interfaces in mOR (PDB: 5C1M) (a) and Hyd_high7 (b) active-state X-ray structures. Water molecules are represented as red spheres and water-mediated hydrogen bonds are indicated by yellow dotted lines. c,d, Interhelical (direct and water-mediated) polar interactions in the TMH core region extracted from the mOR (5c1m) (c) and Hyd_high7 (d) active-state X-ray structures viewed perpendicular to the membrane bilayer. Polar interactions are represented as dotted red lines on the backbone representation of the receptors. The designed mutations rewired the inter-TMH polar interaction network by eliminating native polar contacts between TMH3 and TMHs 5–7 while enhancing TMH7 connections with neighbouring TMHs 1, 2 and 6.
Fig. 6 Hyd-high7 is built of a synthetic allosteric interaction network distant from those of natural GPCRs. a,b, Solvent-mediated polar interaction networks at static–switchable TMH interfaces in mOR (PDB: 5C1M) (a) and Hyd_high7 (b) active-state X-ray structures. Water molecules are represented as red spheres and water-mediated hydrogen bonds are indicated by yellow dotted lines. c,d, Interhelical (direct and water-mediated) polar interactions in the TMH core region extracted from the mOR (5c1m) (c) and Hyd_high7 (d) active-state X-ray structures viewed perpendicular to the membrane bilayer. Polar interactions are represented as dotted red lines on the backbone representation of the receptors. The designed mutations rewired the inter-TMH polar interaction network by eliminating native polar contacts between TMH3 and TMHs 5–7 while enhancing TMH7 connections with neighbouring TMHs 1, 2 and 6.CC BY · PubMed Central
Extended Data Fig. 1 Polar interaction networks connecting static and switchable TMHs in the Hyd_high2 design and A2AR. a, Cartoon representation of the Hyd_high2 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high2 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 1 Polar interaction networks connecting static and switchable TMHs in the Hyd_high2 design and A2AR. a, Cartoon representation of the Hyd_high2 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high2 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 2 Polar interaction networks connecting static and switchable TMHs in the Hyd_low2 design and A2AR. a, Cartoon representation of the Hyd_low2 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_low2 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 2 Polar interaction networks connecting static and switchable TMHs in the Hyd_low2 design and A2AR. a, Cartoon representation of the Hyd_low2 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_low2 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 3 Polar interaction networks connecting static and switchable TMHs in the Hyd_high3 design and A2AR. a, Cartoon representation of the Hyd_high3 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high3 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 3 Polar interaction networks connecting static and switchable TMHs in the Hyd_high3 design and A2AR. a, Cartoon representation of the Hyd_high3 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high3 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 4 Polar interaction networks connecting static and switchable TMHs in the Hyd_low3 design and A2AR. a, Cartoon representation of the Hyd_low3 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_low3 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 4 Polar interaction networks connecting static and switchable TMHs in the Hyd_low3 design and A2AR. a, Cartoon representation of the Hyd_low3 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_low3 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 5 Polar interaction networks connecting static and switchable TMHs in the Hyd_high4 design and A2AR. a, Cartoon representation of the Hyd_high4 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high4 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 5 Polar interaction networks connecting static and switchable TMHs in the Hyd_high4 design and A2AR. a, Cartoon representation of the Hyd_high4 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high4 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 6 Polar interaction networks connecting static and switchable TMHs in the Hyd_high5 design and A2AR. a, Cartoon representation of the Hyd_high5 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high5 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 6 Polar interaction networks connecting static and switchable TMHs in the Hyd_high5 design and A2AR. a, Cartoon representation of the Hyd_high5 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high5 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 7 Polar interaction networks connecting static and switchable TMHs in the Hyd_high1 design and A2AR. a, Cartoon representation of the Hyd_high1 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high1 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.
Extended Data Fig. 7 Polar interaction networks connecting static and switchable TMHs in the Hyd_high1 design and A2AR. a, Cartoon representation of the Hyd_high1 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high1 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation.CC BY · PubMed Central
Extended Data Fig. 8 Polar interaction networks connecting static and switchable TMHs in the Hyd_high6 design and A2AR. a, Cartoon representation of the Hyd_high6 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high6 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation. Y315 and E316 belong to the G-protein Gαs.
Extended Data Fig. 8 Polar interaction networks connecting static and switchable TMHs in the Hyd_high6 design and A2AR. a, Cartoon representation of the Hyd_high6 receptor active state model where the adenosine ligand and side-chains in the vicinity of the designed mutations are highlighted in sticks. b,c, zoomed view on the designed region in Hyd_high6 (b) and A2AR (c). Polar contacts are represented with red dotted lines. Residues are numbered using the Ballesteros Weinstein notation. Y315 and E316 belong to the G-protein Gαs.CC BY · PubMed Central
Extended Data Fig. 9 Fluorescent Thermal Stability Assay of T4Lcp- Hyd_high constructs. Measurements were performed for N-terminus circular permutant T4-Lyzozyme - designed Hyd_high 9 (green) and 7 (red) crystallization constructs bound to the ligand agonist CGS21680. Source data
Extended Data Fig. 9 Fluorescent Thermal Stability Assay of T4Lcp- Hyd_high constructs. Measurements were performed for N-terminus circular permutant T4-Lyzozyme - designed Hyd_high 9 (green) and 7 (red) crystallization constructs bound to the ligand agonist CGS21680. Source dataCC BY · PubMed Central
Extended Data Fig. 10 Structure prediction of the Hyd_high7 design by Alpha-Fold2. a, Superposition between Hyd_high7 X-ray structure and Alpha-Fold2 model (AF2). b, AF2 model colored according to pLDDT score. c, pAE plot. d, e, Structural differences between the models generated by SPaDES or AF2 and the Hyd_high7 X-ray structure (provided as mean ± std calculated over 10 lowest energy models). d, Cα RMSD over the transmembrane region. e, RMSD of designed residue side-chains after local alignment of surrounding 6 residues. Source data
Extended Data Fig. 10 Structure prediction of the Hyd_high7 design by Alpha-Fold2. a, Superposition between Hyd_high7 X-ray structure and Alpha-Fold2 model (AF2). b, AF2 model colored according to pLDDT score. c, pAE plot. d, e, Structural differences between the models generated by SPaDES or AF2 and the Hyd_high7 X-ray structure (provided as mean ± std calculated over 10 lowest energy models). d, Cα RMSD over the transmembrane region. e, RMSD of designed residue side-chains after local alignment of surrounding 6 residues. Source dataCC BY · PubMed Central

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Metadata

Authors
Chen, K.-Y. M., Lai, J. K., Rudden, L. S. P., Wang, J., Russell, A. M., Conners, K., Rutter, M. E., Condon, B., Tung, F., Kodandapani, L., Chau, B., Zhao, X., Benach, J., Baker, K., Hembre, E. J., & Barth, P.
Affiliations
  • École Polytechnique Fédérale de Lausanne (CH)
  • Baylor College of Medicine (US)
  • AbbVie (United States) (US)
  • Eli Lilly (United States) (US)
Corresponding author
Barth, P.
ORCID
Venue
Nature Chemistry
Publisher
Springer Science and Business Media LLC
Published
2025-01-23
Volume
17
Issue
3
Pages
429-438
ISSN
  • 1755-4330 (ISSN-L)
  • 1755-4349
DOI
10.1038/s41557-024-01719-2
PMC
PMC11882447
OpenAlex
W4406768741
Cited by
14
References
3
Citations by year
2026: 9 · 2025: 5
FWCI
4.21
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Chemistry (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Infoscience (Ecole Polytechnique Fédérale de Lausanne) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences
  • Eli Lilly and Company
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
Keywords
cell surface receptors, G protein-coupled receptors, allosteric networks, protein-solvent interactions, protein design, thermal stability, protein-ligand interactions
MeSH terms
Allosteric Regulation, Humans, Ligands, Models, Molecular, Receptors, Cell Surface, Solvents, Protein Engineering, Signal Transduction

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