Chris Kloxin, University of Delaware
Aline Miller, Manchester BIOGEL
Jacek Wychowaniec, University College Dublin
Xuehai Yan, Institute of Process Engineering, Chinese Academy of Sciences
Royal Society of Chemistry Scientific Meetings Grant (S19-0365)
University of Delaware, Materials Science and Engineering
SM09.01: Assembly Rules of Responsive Peptide and Protein based Materials
Friday PM, April 23, 2021
8:00 AM - *SM09.01.01
Socially Distant at the Nanoscale—It’s a (Soft) Matter of Heterochirality
Università degli Studi di Trieste1Show Abstract
Diphenylalanine (Phe-Phe) is a popular building block in nanotechnology and materials science. This motif derives from the sequence of Amyloid beta peptide that is prone to self-aggregation and it is associated to Alzheimer’s disease. Phe-Phe is well-known to self-assemble into nanotubes with an inner water-channel, but their uncontrolled growth results into hierarchical assembly into toxic microtubes. In this work, we show that the hydrophobic inter-molecular (social) interactions are responsible for the association into microtubes, and their replacement with intra-molecular (asocial) contacts is key to avoid microtube formation and alleviate cytotoxicity in vitro.
Interestingly, substitution of one amino acid with its mirror-image to yield D-Phe-L-Phe is sufficient to hinder microtube formation. As a result, a homogenous population of 4-nm wide fibrils is obtained that form a network yielding a macroscopic hydrogel. Full-atom molecular dynamics simulations, single-crystal XRD data and spectroscopic analyses allowed us to unravel the fine (supra)molecular details of
the process. Furthermore, halogenation of the building blocks is an additional variable proved to be effective to fine-tune self-organization.
The soft matter formed by self-assembly of short, heterochiral peptides is reversible and gel/sol transitions can be triggered by a variety of stimuli, including not only temperature and pH, but also light for instance. The talk will provide examples of the recent research avenues undertaken on using heterochirality as a design strategy to achieve supramolecular soft matter to serve different functions.
S.Kralj et al. ACS Nano 2020 doi:10.1021/acsnano.0c06041
8:25 AM - SM09.01.02
Ultra-Short Ionic-Complementary Constrained Peptides (UICPs) as a Chemical Platform for the Development of Bioinspired Multifunctional Nanofibrous Materials
Mohamed Elsawy1,Joseph Hayes2,Jacek Wychowaniec3
De Montfort University1,University of Central Lancashire2,University College Dublin3Show Abstract
Peptide non-covalent assembly has been classically studied as the main culprit implicated in a variety of ‘protein aggregation’ diseases, such as Alzheimer’s, Parkinson’s, Huntington’s, Prion diseases, and others. In the last three decades, the process has attracted material designers as a bioinspired strategy for the development of peptide-based assembling functional nanomaterials; which showed great potential for a wide variety of biomedical and pharmaceutical applications (X. Zhao & S. Zhang. Macromol Biosci. 2007, 7, 13; R.V. Ulijn & A.M. Smith. Chem. Soc. Rev. 2008, 37, 664; A.L. Boyle & D.N. Woolfson. Chem. Soc. Rev. 2011, 40, 4295; A. Altunbas & D.J. Pochan. Top Curr Chem. 2012, 310, 135; Dasgupta et al. RSC Advances 2013, 3, 9117; C. Edwards-Gayle & I. Hamley. Org. Biomol. Chem. 2017, 15, 5867). The high versatility of material requirements for different applications makes it important to have a tuneable and multifunctional system by molecular design.
Recent endeavours in our group focus on the molecular engineering of Ultra-short Ionic-complementary Constrained Peptides (UICPs) as a chemical platform for the development of bioinspired cost-effective multifunctional materials. Using bottom-up rational design approach combined with computational modelling, we have developed the parent UICP tetrapeptide sequence Phg4, which is the shortest reported ionic-complementary peptide that self-assemble into thermodynamically stable β-sheet structures forming amphiphilic nanofibers capable of both gelation and emulsification (J. Wychowaniec et al. Biomacromolecules 2020, 21, 2670). Phg4 nanofibers demonstrated unique surface activity in the presence of immiscible oils and were superior to commercial emulsifiers in stabilizing emulsions and emulgels (EMGs) under a range of storage and stress conditions. These results imply the considerable potential of UICPs as biocompatible excipients in pharmaceutical, cosmetics, and food industries. In addition, interfacial self-assembly has led to the fabrication of nanofibrillar microspheres suitable for cell microencapsulation and scaffolding for supporting cells proliferation and differentiation processes. UICP microspheres were also manipulated for fine-tuning release kinetics of multiple drug cargos, both of hydrophobic and hydrophilic nature.
We are currently developing a range of next generation UICP sequences with a variety of aromatic residue designs and charged residues distributions, for further adjusting molecular assembly, gelation and interfacial properties to meet the intended application needs.
The authors would like to thank International Newton Fund (Newton-Mosharafa Scheme) and the Engineering and Physical Sciences Research Council (EPSRC Grant no: EP/G03737X/1) for financial support of this work, and Diamond Light Source for the Beam Time Award (SM17102). Authors would like to thank Prof. Alberto Saiani from School of Materials, University of Manchester for scientific discussions of SAXS data analysis.
8:40 AM - *SM09.01.03
Harnessing Biological Organization Principles to Engineer Active and Dynamic Materials
The University of Nottingham1Show Abstract
Living systems have evolved to grow and heal through self-assembling processes capable of organizing a wide variety of molecular building-blocks at multiple size scales. While advances in nanotechnology and biofabrication are enhancing our capacity to emulate features of some of these biological structures, it is increasingly evident that recreation of their complexity and adaptability will require new ways to build with molecules such as peptides and proteins. This talk will present our laboratory’s efforts to combine supramolecular events found in nature such as disorder-to-order transitions(1,2), diffusion-reaction processes(2,3), and organic-inorganic interactions(1) with engineering (e.g. biofabrication techniques)(4) or materials science (e.g. host-guest complexes)(5) approaches to organize peptides and proteins into hierarchical and functional materials with emergent properties.
1. Elsharkawy et al (2018). Nature Communications, 10.1038/s41467-018-04319-0.
2. Wu et al (2020). Nature Communications, 10.1038/s41467-020-14716-z.
3. Inostroza-Brito et al (2015). Nature Chemistry, 10.1038/nchem.2349.
4. Hedegaard and Mata (2020). Biofabrication, 10.1088/1758-5090/ab84cb
5. Redondo-Gomez et al (2020). Biomacromolecules, 10.1021/acs.biomac.9b00224.
9:05 AM - SM09.01.04
Protein-Based Conductive Polymers—From Flexible and Stretchable Electronics to a Platform for Light-Stimuli-Responsive Material
Technion–Israel Institute of Technology1Show Abstract
We are inspired by nature for its utilization of proteins for a variety of function, and specifically to our work, the ability of proteins to form high-hierarchical structures and the ability of proteins to mediate charges (electrons, protons and ions) across specific pathways from the nm-scale up to the μm scale. and we use proteins for the formation of responsive materials. With this biological inspiration, we report here on a new family of conductive and free-standing biological materials, where we use different types of proteins as building blocks to form various types of materials. With this in mind, we focus only on proteins that can be produced in bulk quantities and in low cost from raw materials, in which most of our work to date has been focused on the bovine serum albumin protein. We show that using our (bio-)polymerization approach we can form highly elastic polymers in large scale, capable of stretching more than 5 times their length. Due to the relatively high water uptake of our protein-based polymers and the presence of many amino acids residues that can participate in hydrogen bonding, our new protein-based polymers showing good protonic and ionic conductivity. Following the formation of the biopolymer, we show that it can be further functionalized in different ways. For enhanced protonic conductivity we add oxo-acids to the polymer, resulting in measured ionic conduction of >10 mS/cm at room temperature. For enhanced electronic conductivity we can dope the formed polymer with natural electron mediating small molecules. For gaining new light-stimuli-responsive we attach to the protein-based polymer light-responsive molecules, resulting in the large light attenuation of its electrical properties. From blue skies research perspective, the protein-based nature of our materials enables us to explore the governing factors and mechanisms of long-range biological charge transport. Nonetheless, our new protein-based biopolymers have several attractive properties for their possible integration in various applications. Our materials are environmentally friendly, they possess inherent biodegradability and biocompatibility, they have attractive mechanical propeties and their formation obeys to most principals of green chemistry. From a practical point of view, we introduce here a very easy polymerization method that requires no synthesis and it is energy efficient, and in addition, our chosen proteins are having low price tag, resulting in a materials cost of around $1/cm2. Currently, our main targeted application for our new family of materials is for biological interfaces, while other lines of applications include the use of our biopolymers for biomedical application (tissue engineering) and for energy applications such as membranes for fuel cells.
9:20 AM - SM09.01.05
Late News: Artificial Protein Design Rules to Harness Protein Tertiary Structures for Polymeric Materials with Exotic Mechanical Behaviors
The University of Arizona1Show Abstract
Unprecedented physical, chemical, and biological properties of natural materials inspire next-generation polymeric materials for healthcare, defense, and industrial applications. For example, muscle tissues and red blood cells (RBC) largely alter their structures under mechanical stresses to dissipate energy and prevent tissue or cellular damage before recovering when environmental stresses decrease. Single molecule studies revealed that the macroscopic mechanical behaviors are strongly correlated to exotic nanomechanics from their protein tertiary structures. Design rules are in place to guide the incorporation of intrinsically disordered proteins into polymeric materials, which have been established using synthetic polymers. However, design rules to harness protein tertiary structures into macroscopic polymeric materials are unclear because the strand flexibility with structured proteins is significantly dissimilar with nonstructured polymer-based strands.
Here, we designed artificial proteins, composed of rigid tertiary and flexible nonstructured protein modules in specific ratios and arrangements to identify design rules for incorporating protein tertiary structures in polymeric materials. The rigid module consists of RBC ankyrin repeats, known for its solenoidal shape and nanospring behavior under applied external forces. The flexible module comprises polyelectrolytic, nonstructured protein repeats. These protein modules were genetically inserted into a telechelic, associative construct with self-oligomerizing protein endblocks to form hydrogels in aqueous conditions. Using mechanical testing, we identified that an asymmetric flexible-rigid protein design with an optimal flexible length enhances the hydrogel rheological properties compared to hydrogels composed of only flexible or rigid protein modules. This indicates that controlling strand flexibility can improve the crosslinking effectiveness in polymeric materials, which can potentially enhance the macroscopic material performance. Furthermore, our current effort to identify specific and strong self-oligomerizing protein endblocks that will properly exhibit protein nanomechanics at the macroscopic material level will be discussed. This design discovery will culminate in an artificial protein platform that harnesses mechanical proteins with diverse tertiary structures into self-assembled materials for exotic mechanical behaviors, which we expect to advance a wide variety of healthcare applications, including but not limited to tissue engineering, drug delivery, and regenerative medicine.
9:35 AM - SM09.01.07
A Protein-Based Free-Standing and Proton-Conducting Transparent Elastomer as a Sustainable Material for Large Scale Sensing Applications
Technion–Israel Institute of Technology1Show Abstract
A most important endeavor in modern materials research is shifting toward green environmental and sustainable materials. Natural resources are one of the attractive sustainable building-blocks for making environmentally-friendly materials. However, in most cases, the performance of nature-derived materials does not match the performance of carefully designed synthetic materials, particularly for conductive polymers, which is the topic here. Inspired by the natural role of proteins in mediating protons, we show here their utilization for making free-standing transparent polymer having highly-elastic nature and proton conductivity comparable to synthetic polymers. Importantly, the polymerization process is relying on the natural protein crosslinkers and is spontaneous and energy-efficient. The used protein, bovine serum albumin, is one of the most affordable proteins, resulting in the ability to form large-scale materials at low cost. Due to the inherent biodegradability and biocompatibility of the elastomer, it is promising for biomedical application, and here we show its immediate utilization as a solid-state electrode for sensing physiological signals.
9:39 AM - SM09.01.08
Late News: Artificial Protein Design as an Effective Material Platform for Antimicrobial Peptides
Fathima Doole1,Lauren Melcher1,Christopher Camp1,Anne Wertheimer1,Abhishek Singharoy2,Michael Brown1,Minkyu Kim1
University of Arizona1,Arizona State University2Show Abstract
Antimicrobial peptides (AMPs) are a promising solution to combat antibiotic-resistant superbugs. The major drawbacks of AMPs are their rapid in vivo degradation and renal filtration which have led to poor AMP efficacy. Anchoring AMPs with synthetic polymer tethers on biomaterial surfaces can overcome these obstacles by enhancing AMP stability and protecting AMPs from renal filtration. Further, modulating the physicochemical properties of synthetic polymer tethers can potentially maximize AMP efficacy. However, intricate material synthesis and inconsistent yields of tethering AMPs on material surfaces result in varying material performance.
Using artificial protein technology, we developed an “All-in-One” (AiO) protein-based antimicrobial material that contains three major components, similar to conventional AMP-incorporated materials: a material scaffold, biopolymer tether, and an AMP. An elastin-like protein (ELP) was designed as the material scaffold that when in aqueous solution under physiological temperature will make micelles or the ELP can be photo-crosslinked to make hydrogels. Next, a hydrophilic, flexible protein sequence was introduced to the material scaffold to act as a biopolymer tether. Finally, two of the most common AMPs, LL37 and Pexiganan, were chosen to demonstrate and evaluate AiO protein-based antimicrobial materials. To investigate the role of the biopolymer tether, AiO antimicrobial materials with and without tethers were biosynthesized. The AiO material with the tether reduced the growth of Staphylococcus aureus, gram-positive bacteria, by 50% more than the material with non-tethered AMP. This could be due to greater flexibility and degrees of freedom of tethered AMPs, which can effectively interact with the bacteria. To investigate this hypothesis, all-atom molecular dynamics simulations are being employed to understand the atomistic level details of the biopolymer tether on AMP activity and membrane interaction. Artificial AiO protein development provides consistent yields and materials performance, as well as the opportunity to engineer material components at a single amino acid level precision to improve the AMP efficacy. Therefore, we anticipate that the AiO protein will advance the use of diverse AMPs as an effective and wide-reaching therapeutic strategy to mitigate antimicrobial resistance
9:43 AM - SM09.01.09
Role of Calcium Signaling on Regulated Cell Death Induced by Membrane-Interacting Peptide Amphiphiles
Manal Binqabbus1,Damien Samways1,Shantanu Sur1,Samuel Stupp2,Morgan Reynolds1,Michael Sanborn1
Clarkson University1,Northwestern University2Show Abstract
Peptide amphiphiles (PA) consist of short peptide chains linked to an alkyl tail that can undergo self-assembly into nanofibers. Previous studies have shown that exposure to many types of PA results in cell death, and the assumption has been that this is primarily due to disruption of cell plasma membrane integrity. However, preliminary data in our laboratory has indicated that the effects of certain weak beta-sheet forming cationic PAs on inducing cell death might involve specific stimulation of signal transduction pathways rather than coarse disruption of biological membranes. Our goal was to investigate the effect of PA exposure on intracellular Ca2+ homeostasis, which is known to be a major regulator of cell survival. Utilizing live cell Ca2+ imaging with the Ca2+ indicator Fluo-4, we found that exposure to PA stimulated slow sustained elevations in intracellular Ca2+ concentration ([Ca2+]i). This PA-induced [Ca2+]i persisted in the absence of added extracellular Ca2+ ruling out the possibility that it was due to Ca2+ leak across a disrupted plasma membrane. Rather, the elevation in [Ca2+]i appears consistent with the mobilization of Ca2+ from the intracellular endoplasmic reticulum stores, in part through a phospholipase C (PLC)-dependent signal transduction pathway. We hypothesized that the PA-dependent elevation in [Ca2+]i marked the first step in the observed PA-induced cell death. However, surprisingly the cytotoxicity of PA was not significantly reduced by either PLC inhibition or by suppressing Ca2+ elevations in the cervical cancer cells by pre-incubating them with the intracellularly active Ca2+ chelator, BAPTA-AM. While PA-induced changes in [Ca2+]i do not appear to cause the subsequent PA-associated cell death, these data nevertheless support the hypothesis that PAs can influence vital intracellular singling processes.
9:47 AM - SM09.01.10
Identifying New Strategies to Promoted Adhesion to Non-Polar Substrates by Bacterial Surface Display
Mark Kozlowski1,Joshua Orlicki1,Randall Hughes1,Thomas Segall-Shapiro1,Randi Pullen1,Jimmy Gollihar1
U.S. Army Research Laboratory1Show Abstract
Polystyrene (PS) and polypropylene (PP) are ubiquitous plastics in our modern world. However, their highly non-polar characteristics can lead to difficulty forming a good adhesive bond. Current methods for PS and PP adhesion rely on high temperatures, solvent plasticization, or extensive pre-treatment and priming. Identifying new strategies to enable good adhesive bonding would provide opportunities for facile repair in the field, new designs for capacitors, and the creation of new types of composite materials.
Biology may point the way to these new strategies for chemically-based adhesion of non-polar substrates. For example, the filamentous projections (hyphae) of fungi must breach an air-water interface when colonizing new environments and seeking nutrients, and do so by the secretion of a family of natural surfactants known as hydrophobins . Hydrophobins have already been shown to adhere to highly non-polar surfaces, such as Teflon , and there is at least one previous report of a designed chimeric hydrophobin successfully adhering to polystyrene , meaning this is a promising class of materials deserving further exploration.
Screening the chemical compositional space afforded by peptides and the natural amino acids would be nearly impossible if undertaken in a serial fashion. Recently, the Army Research Laboratory developed a peptide surface display and high-throughput library screening system to find candidate peptides capable of adhering to polylactic acid , and to gold nanoparticles . The on-cell peptide screening has the advantages of easy recoverability, the ability to propagate and sequence the genetic code of the adhesive peptides, and possible further improvements of the peptides by directed evolution. In the present work, we use these previously-developed surface display methodology, a peptide library, and a separate hydrophobin library, to screen for affinity and (hopefully) adhesion to non-polar plastics. We believe this work will encourage further exploration of biologically-inspired adhesive agents for non-polar plastics, and provide key insights for the chemical synthesis of new types of non-polar material adhesives.
 P.W. Cox, P. Hooley, Hydrophobins: New prospects for biotechnology, Fungal Biology Reviews 23(1) (2009) 40-47.
 M. Linder, G.R. Szilvay, T. Nakari-Setälä, H. Söderlund, M. Penttilä, Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei, Protein Sci 11(9) (2002) 2257-2266.
 I. Sorrentino, M. Gargano, A. Ricciardelli, E. Parrilli, C. Buonocore, D. de Pascale, P. Giardina, A. Piscitelli, Development of anti-bacterial surfaces using a hydrophobin chimeric protein, International Journal of Biological Macromolecules 164 (2020) 2293-2300.
 S.D. Stellwagen, D.A. Sarkes, B.L. Adams, M.A. Hunt, R.L. Renberg, M.M. Hurley, D.N. Stratis-Cullum, The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries, BMC Biotechnol. 19(1) (2019) 12.
 J.P. Jahnke, H. Dong, D.A. Sarkes, J.J. Sumner, D.N. Stratis-Cullum, M.M. Hurley, Peptide-mediated binding of gold nanoparticles to E. coli for enhanced microbial fuel cell power generation, MRS Commun. 9(3) (2019) 904-909.
Distribution Statement A: Approved for public release: distribution unlimited.
9:51 AM - SM09.01.11
Late News: A Peptide Based Latent Crosslinker Activated by Thiol-Thioester Exchange Reaction
Makafui Folikumah1,2,Marc Behl1,Andreas Lendlein1,2
Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Geesthacht1,University of Potsdam2Show Abstract
Thiol-thioester exchange (TTE) reaction can occur smoothly in neutral aqueous media and is therefore worthwhile to be considered as potential dynamic covalent chemistry for physiological environments [1-2]. Typically, the thiol and the thioester functionalities reacting here are part of two separate molecules. Systems making use of a single molecule providing both functional groups employ hydrophobic aromatic moieties. In these cases, a catalyst might be required in order to control the direction of the equilibrium .
The hydrophobicity of these substrates and the liberation of foul-smelling aromatic thiols limit their use for biomedical application. Here we report on a thio-depsipeptide (TDP), Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH synthesized by the modification of a standard collagenase activity peptide, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-OEt. TDP was capable of a ‘pseudo’ intramolecular TTE reaction to yield α, ω-free thiol bearing peptide crosslinker without perceivable foul-smell. We could demonstrate in electrospray ionization mass spectrometry studies that the TDP, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH, self-generates a dithiol peptide crosslinker, HSLeu-Leu-Gly-NEtSH (BTDP) as a TTE product in aqueous medium in addition to Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtS-Gly-Leu-AcPro (TXP). When mixed with a panel of thiols, cysteine (pKa 10.8), glutathione (pKa 9.6), methylthioglycolate (pKa 7.8), 4-mercaptobenzoic (pKa 5.9), and para-nitrophenol (pKa 4.6) in equimolar concentrations, the fate of exchange products between TDP and the external thiols was found to be dependent on the relative pKa of these thiols to the attacking TDP thiol. L-cysteine with similar thiol pKa as the attacking TDP thiol yields an additional exchange product Ac-Pro-Leu-Gly-SCys-OH, with a corresponding increase in BTDP peak intensity than observed for TDP only. 4-mercaptobenzoic and para-nitrophenol with more acidic thiols relative to the attacking TDP thiol however resulted in a decrease of BTDP peak intensity caused by the lack of breakdown of their respective tetrahedral intermediates, which were clearly visible in the recorded spectra.
A concept for the design of water-soluble single TTE substrates without the need for incorporation of acidic aromatic thiols is presented. Since aromatic thiols are known for their toxicity and unpleasant smell after TTE reactions the newly developed TDP could potentially enable in situ bioconjugation and crosslinking applications.
 M.G. Woll and Gellman, S. H., J. Am. Chem. Soc., 126 (36), 11172-11174, 2004
 R. Larsson, Z. Pei, O. Ramström, Angew. Chem. Int. Ed., 43 (28), 3716-3718, 2004
 C. Wang, S. Mavila, B. T. Worrell, W. Xi, T. M. Goldman, C. N. Bowman, ACS Macro Lett, 7 (11), 1312-1316, 2018
SM09.02: Peptide and Protein based Materials: From Assembly to Applications in Biological Context I
Friday PM, April 23, 2021
11:45 AM - *SM09.02.01
β-Sheet Forming Peptide Hydrogels—From Self-Assembly to Functional Biomaterials
The University of Manchester1Show Abstract
The use of non-covalent self-assembly to construct materials has become a prominent strategy in biomaterials science offering practical routes for the construction of increasingly functional materials for a variety of applications ranging from cell culture and tissue engineering to in-vivo cell and drug delivery. A variety of molecular building blocks can be used for this purpose, one such block that has attracted considerable attention in the last 20 years is de-novo designed peptides. The β-sheet motif is of particular interest as short peptides can be designed to form β-sheet rich fibres that entangle and consequently form very stable hydrogels. These hydrogels can be easily functionalised using specific biological signals and can also be made responsive through the use of enzymatic catalysis [3-4] and/or conjugation with responsive polymers . Through the fundamental understanding of the self-assembly and gelation of these peptides across length scales [6-8] we have been able to design hydrogels with tailored properties for a range of applications including for the culture of a variety of cells[9-11], injectable and sprayable hydrogels for cell and drug delivery [12-13] as well as shear thinning hydrogel for 3D bio-printing [14-15]. The intrinsic biocompatibility  and low immunogenicity  of these materials makes them also ideal for TERM applications. Recently we have demonstrated their potential in a range of TERM applications including, oesophagus , nerve , intervertebral disk  and cardiac  regeneration.
1. Zhang, S. G., Nature Biotechnology 2003, 21, 1171; 2. Zelzer, M. et al. Chemical Society Reviews 2010, 39, 3351; 3. Guilbaud J.B. et al. Langmuir2010, 26, 11297; 4. Guilbaud J.B. et al. Biomacromolecules 2013, 14, 1403; 5. Maslovskis A. et al. Langmuir 2014, 30, 10471; 6. Elsawy, M. A. et al., Langmuir 2016, 32, 4917; 7. Gao, J. et al., Biomacromolecules 2017, 18, 826; 8. Wychowaniec, J. Et al. Biomacromolecules 2020, 21, 2285; 9. Mujeeb, A. et al., Acta Biomaterialia 2013, 9, 4609-4617; 10. Castillo Diaz, L. A. et al., Journal of Tissue Engineering 2014, 5, 2041731414539344; 11. Castillo Diaz, L. A. et al. Journal of Tissue Engineering 2016, 7, 2041731416649789; 12. Roberts, D. et al. Langmuir 2012, 28, 16196; 13. Tang, C. et al., International Journal of Pharmaceutics 2014, 465, 427; 14. Raphael, B. et al., Materials Letters 2017, 190, 103; 15. Chiesa J. Et al. Frontiers in Medical Technology DOI:10.3389/fmedt.2020.571626 (2020); 16. Morris O. et al. Journal of Labelled Compounds and Radiopharmaceuticals 2017, 60, 481; 17. Markey A. et al. Journal of Peptide Science 2017, 23, 148; 18. Kumar D. et al. Advanced Functional Materials 2017, 27, 1702424; 19. Faroni et al. Advanced Healthcare Materials, 2019, 1900410; 20. Corimo L. et al. Acta Biomaterialia 2019, 92, 92; 21. Burgess K. et al. Materials Science & Engineering C 2020 in press.
12:10 PM - SM09.02.02
Simulating Peptide Self-Assembly on Single Layer Materials via Asynchronous Markov Chain Algorithms
Siddharth Rath1,Michael Malone1,Chandler King1,Mehmet Sarikaya1
University of Washington1Show Abstract
Predictively controlling the self-assembly of biomolecules at solid interfaces is crucial for the development of functional substrates at bio/nano soft interfaces. The self-assembly of peptides is largely impacted by environmental conditions such as temperature, pH, and concentration. However, the extent to which these environmental conditions dictate peptide self-assembly on atomically single-layer materials is largely unknown. Here we show a computational modeling approach that enables a greater understanding of how adjustments of environmental conditions affect peptide self-assembly. We modify asynchronous Markov chain algorithms to investigate the impact of environmental conditions on peptide self-assembly. The original purpose of the Markov model was to visualize programmable matter that can achieve complex self-organizing ensembles. This is carried out using particles with extremely limited computational power. We modify these algorithms to achieve a virtual self-organizing particle system representative of self-organizing solid binding peptides. The overall self-assembly process is simplified to include the most significant steps: 1. Peptide adsorption/desorption, 2. Diffusion across substrate, 3. Clustering of peptides, and 4. Break-up of clusters into linear formations based on favorable conformation-mediated intermolecular interactions. Our Markov model currently allows for variable adjustments in particle surface concentration, adsorption rate, strength of intermolecular interactions, and diffusion coefficients. Various combinations of these bias parameters yield unique self-organizing particle systems with distinct characteristics, similar to the differences in self-assembly characteristics between various solid binding peptides. We show, therefore, that Asynchronous Markov Models provide a simple modeling platform for biomolecular self-assembly on atomically flat solids along with avenues to tune binding energies, absorption/desorption rates, temperature, effects of concentration, pH, and structure-mediated intermolecular interaction energies as well as lattice mismatch parameters. Such models allow one to fit simulated models with AFM images, including grain size, chiral angles, degree of ordering, and surface coverage, estimate several energetic parameters involved in the self-assembly process, towards better characterization and understanding of interfacial properties in soft bio/nano interfaces and their implementation into practical devices. The research is supported by NSF-DMREF program.
12:20 PM - *SM09.02.03
Programming Sequence-Defined Peptoids for Bio-Inspired Synthesis of Functional Nanomaterials
Pacific Northwest National Laboratory1Show Abstract
Proteins are the molecular machines that carry out the vast array of functions needed for the survival and propagation of all cellular organisms. Many proteins form this machinery by folding into functional building blocks that self-assemble into extended networks to deliver sequence-specific functions ranging from photosynthesis, to molecular separation, selective ion transport, and tissue mineralization. Inspired by nature, many sequence-defined molecules have been exploited for the preparation of nanostructured functional materials. Peptoids are one of the most advanced classes of sequence-defined synthetic foldamers. By bridging the properties of proteins and polymers, they offer unique opportunities for the synthesis of biomimetic materials with controlled structures and tunable functions.
In this talk, I will discuss our recent efforts in designing amphiphilic peptoid sequences for bio-inspired synthesis of functional materials (e.g. membrane-mimetic 2D nanosheets, dynamic nanotubes, pore-forming networks, and flower-like fluorinated nanoparticles). For these self-assembly systems, peptoid-peptoid and peptoid-substrate interactions play critical roles in the peptoid assembly and can be tuned through the peptoid sidechain chemistry. By programming these peptoids with responsive functional groups and sidechains, we developed a variety of responsive hierarchical nanomaterials. Due to the high stability and programmability of these biomimetic materials, we further demonstrated the incorporation of various functional groups into these peptoid-based materials for specific applications. Our results indicate that self-assembly of amphiphilic peptoids into hierarchical structures can be used as a robust platform to develop biomimetic materials with tunable structures and controllable functions.
12:45 PM - SM09.02.04
Designed Interfaces Between Proteins and Inorganic Crystals for Templated Assembly and Co-Assembly
Pacific Northwest National Laboratory1Show Abstract
Previously we have shown we could use Rosetta to design proteins that exhibited a lattice match to mineral surfaces. We discovered that we could exploit those interactions and designed protein-protein interfaces to generate a variety of ordered 2D phases (micrometer-long wires and extensive honeycomb arrays) that were strongly dependent on electrolyte type, electrolyte and protein concentration, and protein sequence. Even without designed protein-protein interfaces, rod shaped proteins with the protein-mineral interface assembled into 2D ordered phase with defined planes characteristic of smectic phases, which is surprising considering that 2D smectic phases have not been observed in colloidal nanorod systems nor are they predicted by Monte Carlo simulations of non-interacting rods. Thus, these assemblies must result from the competition between protein-protein interactions, protein-mineral interactions, and colloidal forces. Our knowledge of the protein-mineral interactions is informed by machine learning analysis that shows the orientation dependent energy landscape is complex and depends on electrolyte type and concentration, and theoretical analysis that shows a far-field reversal in the polarization response which may be the cause the distinct behavior seen with Na+ vs K+. We are now exploring the co-assembly of different proteins at a solid-liquid interface to both investigate the role of complementarity and frustration in defining order and to generate higher-order assemblies. In addition, we are working to “lock” the protein assemblies formed in one set of conditions as a means of creating extensive, anisotropic 2D protein layers as scaffolds for subsequent mineralization and 2nd component assembly under disparate conditions.
12:55 PM - SM09.02.05
Understanding the Impact of Sequence Length, Composition, and Dispersity on the Melting Transition and Assembly of Collagen-Like-Peptide (CLP) Triple Helices
Phillip Taylor1,April Kloxin1,Arthi Jayaraman1
University of Delaware1Show Abstract
Recent advances in materials design, synthesis, and simulation have allowed the creation of biomimetic materials with responsive yet controllable physicochemical properties. Materials that self-assemble into desired morphologies such as fibrils and supramolecular networks are of particular interest, where their ability to self-assemble can be tuned by applying external stimuli such as heat, light, pH, and salt for a range of applications including in drug delivery and tissue engineering. In this talk, we will present our recent work involving coarse-grained (CG) simulation studies on the melting transitions, fibrillar assembly, and gelation of collagen-like peptides (CLPs). CLPs are thermoresponsive biopolymers in which each CLP chain is made up of repeat units of amino acid triplets, (X-Y-G), where X and Y are usually proline (P) and hydroxyproline (O), respectively. Like native collagen, CLPs have been shown to assemble to form triple helices, fibrils, and gels in aqueous solutions thus exhibiting self-assembly at multiple length scales. In this work, we extend our CG CLP model to simulate CLP heterotrimers in which the length of each of the three CLP strands forming the triple helix can be different. Inspired by the heterotrimeric nature of natural collagens, we investigate CLP heterotrimers with sticky ends which self-assemble to form fibrils and fibrillar networks driven by interchain hydrogen bonding. We explore how various design parameters including the length and number of sticky ends in each CLP triple helix, CLP triple helix concentration, and temperature (above and below CLP melting transition) impact CLP assembly. Overall, our work highlights the predictive capabilities of MD simulations in guiding experiments, as these complex peptide systems with unique molecular insights can inform new system designs and streamline the discovery of new, biomimetic platforms.
1:05 PM - *SM09.02.06
Stimuli Responsive Protein Vesicles for Biocatalysis and Drug Delivery
Georgia Institute of Technology1Show Abstract
Protein vesicles incorporating functional, globular proteins have potential in a number of bio-applications such as drug delivery, biocatalysis, and sensing. We have previously created protein vesicles from mCherry-zipper-ELP protein complexes where ELP is a thermo-responsive elastin-like polypeptide, zipper is a coiled-coil, and mCherry is a model folded protein. As we utilize these vesicles, we have replaced mCherry with more useful functional proteins and have engineered the vesicles to provide both stability and responsiveness. We implemented non-natural amino acid incorporation to enable photocrosslinking strategies to stabilize vesicles and control their swelling and release of cargo as a function of salt concentration. We have modified the ELP amino acid sequence to create vesicles that are pH sensitive and swell or disassemble at acidic pH. With this information, we have demonstrated assembly of biocatalytic vesicles with significant improvements in activity over soluble enzyme and produced vesicles for drug delivery capable of carrying and releasing therapeutic cargoes. The wide range of vesicle properties and functions exhibited in these examples, highlight the versatility of protein vesicles as functional and responsive protein materials.
1:30 PM - SM09.02.07
Controlling Blood Coagulation in Supramolecular Vascular Access Grafts via a Feedback-Response Mechanism
Boris Arts1,2,Patricia Dankers1,2
Technische Universiteit Eindhoven1,Institute for Complex Molecular Systems2Show Abstract
Currently 1 in 1000 people in Europe suffer from end-stage renal disease and require hemodialysis1. Hemodialysis is a medical procedure to remove waste products from the blood where the blood circulation of the patient is directly connected to a dialysis machine, often, via a vascular access graft (VAG). The primary failure of VAGs are due to its low patency rate. Several studies showed that coating the inner lining of the graft, e.g. with heparin, improves the patency rate2. However major concerns are expressed regarding the long-term efficacy of those coatings. Hereto the aim of this study is to improve the hemocompatibility of these grafts by controlling blood coagulation through a feedback-response mechanism.
Here we make use of bisurea (BU)-based supramolecular polymers. BU motifs can self-assemble via hydrogen bonding resulting in dynamic crosslinks embedded in a soft amorphous polymer phase. Bioactive molecules, e.g. peptides, can be functionalized with BU and mixed in through a modular approach to implement specific properties in the material3.
In our approach heparin is conjugated to the surface of our VAGs via a thrombin cleavable peptide (TCP). When in contact with whole blood the enzyme thrombin is designed to cleave the peptide causing heparin to be released (response). In turn heparin can form an inhibitory complex with anti-thrombin, naturally present in blood, thereby inhibiting thrombin activity and decreasing heparin release (feedback).
TCPs were synthesized by solid phase peptide synthesis. Next, heparin (Hep) was functionalized with TCPs (Hep-TCP). To surface functionalize the graft material an inverse electron demand Diels Alder (iEDDA) reaction was utilized between tetrazine (Tz) and bicyclooctyne (BCN). Hereto BU-Tz motifs were mixed in the material, while TCPs were functionalized with a BCN moiety.
Fibrous scaffolds were produced by electrospinning of the BU-polymer with or without BU-Tz. Scaffold morphology and fiber diameter were assessed by scanning electron microscopy (SEM). The iEDDA reaction was carried out by incubating the scaffold in reaction mixture containing Hep-TCP. Surface functionalization was investigated through x-ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurements, and toluidine blue staining.
Results and discussion
TCPs were successfully synthesized. The grafting density of TCP on heparin was analysed by 1H-NMR and UV/vis spectroscopy. A grafting density of 7 was obtained. Scaffolds were obtained by electrospinning with an average fiber diameter of 0.9 ± 0.1 µm and 1.0 ± 0.1 µm for pristine and BU-Tz containing scaffolds, respectively, as observed by SEM.
Activity of thrombin towards TCP and Hep-TCP was assessed in solution by fluorescence spectroscopy. Michaelis-Menten kinetics was used to determine the Michaelis-Menten constant (Km) and maximum rate of cleavage (Vmax). Both TCP and Hep-TCP showed similar values of Km. However, Vmax almost decreased by a factor 2. This difference might be attributed to inhibitory effects of heparin towards thrombin. Next, scaffolds were surface functionalized with Hep-TCP by incubation in reaction mixture. XPS, WCA and toluidine blue staining revealed the presence of heparin at the surface of the scaffold.
It was shown that heparin could be successfully functionalized at the surface of our supramolecular polymer scaffolds. Next, further investigation towards the cleavage of heparin from the surface of the scaffold should be carried out.
1. Kramer, A. et al. The European Renal Association – European Dialysis and Transplant Association (ERA-EDTA) Registry Annual Report 2015: A summary. Clin. Kidney J. 11, 108–122 (2018).
2. Walpoth, B. H. et al. Improvement of patency rate in heparin-coated small synthetic vascular grafts. Circulation 98, II319-23; discussion II324 (1998).
3. Koevoets, R. A. et al. Molecular recognition in a thermoplastic elastomer. J. Am. Chem. Soc. 127, 2999–3003 (2005).
1:40 PM - SM09.02.08
Late News: Binding Affinity of Oligomers Towards SARS CoV 2 S-Protein Through Machine Learning and Experimental Validation
Craig Neal1,Katalina Biondi1,Elayaraja Kolanthai1,Aida Tayebi1,Niloofar Yousefi1,Ganesh Balasubramanian2,Ozlem Ozmen1,Ivan Garibay1,Sudipta Seal1
University of Central Florida1,Lehigh University2Show Abstract
The COVID-19 pandemic has led to over 80 million people becoming infected and has claimed more than 1.7 million lives worldwide. As the pandemic progresses, it has become evident that long term solutions for what can be considered as “new normal” are needed. In this work, we ultimately aim to develop a multi-layer, functional coating architecture to capture-and-kill SARS-CoV-2, the virus responsible for COVID-19, which can be formed over personal protective equipment and high-touch surfaces requiring frequent sanitation such as in airports, airplanes, hospitals, public spaces, etc. To facilitate the initial capture of SARS-CoV-2, a high-specificity binding element must be determined. Given the immediate, dire need to block viral transmission: our approach aims to accelerate the discovery of such components. We use machine learning techniques such as transfer learning to leverage the vast amount of data that exists in drug discovery databases to overcome limitations of data sparsity for COVID-19-related protein/molecular interactions. First, graph convolutional neural network (GCNN) is pre-trained using a dataset curated from public databases such as DrugBank, ChemBL, and DUDE to build a binary classifier. This classifier is used for virtual screening of binding affinities between small molecules (ligands, aptamers) and relevant proteins (targets). Next, we fine-tune the resulting GCNN on COVID-19-related data. This GCNN model automatically extracts features from defined “protein pockets” (i.e. binding regions; e.g. SARS-CoV2 S-protein receptor binding domain site) and molecular graph representations of ligands, and classifies them as inactive or active. We rank candidate ligands based on binding scores using molecular docking studies for test priority/potential efficacy. Umbrella sampling simulations using molecular dynamics are employed to determine the free energy of binding for the protein-ligand complex as well as to examine the hydrogen bonding, hydrophobic and non-bonded interaction energies to identify possible stimulus for enhancing the specificity of the binding element. Finally, higher priority candidates are further evaluated and experimentally validated using a commercial binding (S-protein/ACE2) inhibition based assay.
1:43 PM - SM09.02.09
High Performance Computing and the Covid-19 Virus
Sreejita Patra2,Bhushan Dharmadhikari1,Prabir Patra3,4
MInnesota State University, Mankato1,Fairfield Warde High School2,University of Bridgeport, Bridgeport3,University of Bridgeport4Show Abstract
An effective way to predict the binding sites of SARS-COV-2 proteins, and enzymes in pulmonary surfactant, is by taking advantage of present-day High-Performance Computing (HPC) capabilities. These are able to identify the response of various pulmonary surfactant proteins, as well as enzymes on the Covid-19 spike proteins. The fundamental dynamics of COVID-19 and lung defender proteins; such as, but not limited to, pulmonary surfactant protein D (SP-D); need to be understood at the atomic level in order to develop new target-specific drugs that can fight against the infection. Amongst many computational and bioinformatics tools available today, the Molecular dynamics (MD) simulation is of great importance.
We performed an MD simulation on two pulmonary surfactant proteins: SP-A and SP-D, using NAMD and CHARMM27 force field parameters. Molecules were solvated using the TIP3P water model with 150 mM of sodium and chloride ions. All the parameters of the simulation were set according to the parameters described in Jeong et al., 2020. The goal of our simulation study is to help develop a carbon nanotube-based (CNT) protein sensor, which will detect the ultra-low quantity of pulmonary surfactant protein levels with a potential of lung disease implications. The MD results show higher readings of RMSD and SASA in the case of SP-D and CNT interactions. SP-D considers CNT to be a foreign body, and shows a higher level of protein structure change in comparison to SP-A. This may also be the case in the presence of COVID-19 pathogens, and could be a factor in the elevated levels of SP-D. Performing similar HPC simulations with COVID-19 can give us insight into how the proteins and lipid complex inside the pulmonary surfactant are affected by the deadly pathogen.
SM09.03: Peptide and Protein based Materials: From Assembly to Applications in Biological Context I
Friday PM, April 23, 2021
2:15 PM - *SM09.03.01
Modulating the Temperature Response and Performance of Elastin-Based Adhesives
Purdue University1Show Abstract
Our group has developed bioinspired protein-based adhesives that combine adhesion from DOPA residues found in mussel adhesive proteins with the mechanical properties of elastin, which can also coacervate in response to the environment. These proteins are cytocompatible, provide the strongest bonds of any rationally designed protein when used completely underwater, and can be easily applied underwater because they coacervate in physiological conditions. Recently, we demonstrated that incorporating short, charged peptide tags can be used to tune the coacervation temperature of these proteins. Furthermore, the recombinant protein design allowed us to systematically probe the individual contributions and interactions of DOPA and thiol chemistries to adhesion.
3:05 PM - SM09.03.03
Hierarchically Organized Structure of Electrospun Nanofibers from Computationally Designed Peptide Bundlemers
Kyunghee Kim1,Chris Kloxin1,Jeffery Saven2,Darrin Pochan1
University of Delaware1,University of Pennsylvania2Show Abstract
Fiber materials from natural proteins or synthetic polymers are ubiquitous in technology due to many features including a high aspect ratio, large surface area, structural tunability, and excellent/tunable mechanical properties. In contrast to synthetic polymers, which involve a distribution of chain lengths/molecular weights and random coil structures, proteins and peptides can have defined sequences of amino acids with uniform lengths/molecular weights, allowing for precise control over molecules, supramolecular structures, and overall hierarchical assemblies. Herein, computationally designed peptides were used to assemble coiled-coil, or ‘bundlemer’ building blocks for the construction of higher-order fiber materials. The two different, designed bundlemers are coiled-coil bundles that, subsequent to coiled-coil formation, covalently interact with each other to produce rigid-rod, peptide-based polymers. The extreme rod-like morphology and resultant properties of the bundlemer polymers are confirmed by transmission microscopy (TEM) and rheology. Due to its molecular rigidity, the rod chains can exhibit lyotropic liquid crystalline behavior in concentrated solution. The resultant rod chains are subsequently employed to fabricate fiber materials via electrospinning, preserving their unique orientational behavior within the fibers. The rod chains are preferentially aligned along the fiber axis, which is confirmed by x-ray scattering, scanning microscopy, and polarized optical microscopy. Additionally, the mechanical properties of the final nanofibers will be presented.
3:15 PM - *SM09.03.04
Programming Phase Separation of (Poly)Peptides for Controlled Nano- and Micro-Structured Materials
University of Delaware1Show Abstract
Significant attention has been paid to the sequence specificity of intrinsically disordered peptide and proteins owing to their importance in regulating spatiotemporal organization of membraneless organelles in cells and their demonstrated versatility in producing hydrogels, nanoparticles, and sensor platforms. In our laboratory, we have employed amino acid sequences inspired by structural proteins such as collagen, elastin, and resilin, and have tailored their stimuli-responsive behavior to enable finely tuned control over both microscale and nanoscale structures. Their conjugation via chemical methods affords biomaterials with diverse properties responsive to multiple triggers, and select modification of their sequences facilitates nuanced manipulation of their assembly and responsiveness. We have also investigated the controlled retention and release of cargo via biomimetic mechanisms, offering substantial improvement in activity for both small molecule and macromolecular cargo, with targeted applications in tissue repair.
3:40 PM - *SM09.03.05
Adaptable Hydrogels for Organoid Culture
Stanford University1Show Abstract
The term “organoid” refers to an artificially grown collection of cells that resembles aspects of a native organ. While organoid culture has the potential to revolutionize our understanding of human biology, current protocols rely on the use of Matrigel, a complex, heterogeneous protein-based material with large batch-to-batch variations that hinder reproducibility. In response, several groups have begun designing synthetic hydrogel systems based on polyethylene glycol to enable the reproducible culture of organoids. As a fully biodegradable alternative, here we present the design of an engineered protein hydrogel system for reproducible organoid culture. Our family of double-network hydrogels undergo two stages of crosslinking: the first stage uses reversibly dynamic covalent chemistry bonds, while the second stage reinforces the hydrogel through thermal-induced protein aggregation. Recently, the matrix stress relaxation rate (i.e. the ability of a hydrogel to remodel its network connectivity in response to an applied stress) has been demonstrated to have profound effects on encapsulated cells. To date, the role of matrix stress relaxation on organoid cultures has been underexplored. Our engineered double-network of physical interactions results in a gel with a broad dynamic range of tunable mechanical properties, where the gel stiffness is set by the number of crosslinks and the gel stress relaxation rate is independently set by the kinetics of the crosslink binding and unbinding. These novel, double-network hydrogels are being used to study the role of mechanotransduction in the culture of several different types of patient-derived, human organoids.
4:05 PM - SM09.03.06
Designed 2D Binary Protein Materials Geared to Modulate Cells Behavior
Ariel Ben-Sasson1,Joseph Watson2,Alice Bittleston2,Logeshwaran Somasundaram1,Justin Decarreau1,Andrew Drabek3,Stephen Blacklow3,4,Hannele Ruohola-Baker1,Emmanuel Derivery2,David Baker1
University of Washington1,University of Cambridge2,Harvard Medical School3,Dana-Farber Cancer Institute4Show Abstract
Imposing order onto the fundamentally disordered 2D cell membrane matrix upon introduction of an external cue, enables to modulate and reshape the cell behavior. We recently designed a de novo binary 2D protein co-assembling system1 which, unlike its natural single component counterparts such as S-layers,2,3 rapidly assemble into an ordered hexagonal array only upon mixing of its two distinct building blocks (protein components). In a binary architecture, individual components can be designed to remain soluble and amenable for facile functionalization in ambient conditions, and the assembly onset timing and location can be directed and determined by the experimental setup.4 We leverage these novel properties of the binary 2D material and repurpose the system to specifically interact with cell membrane proteins and modulate their surface distribution and downstream signaling.
To this end we first selected a membrane receptor, genetically- or peptide-fused a compatible ligand to one of the arrays components, saturated the cell membrane receptors with said component, removed unbound components from the cell environment, and triggered arrays formation by introducing the second component into the system. We found that the arrays’ dihedral building blocks (each component monomer first forms a dihedral homooligomer and the mixture of these forms the arrays) that need to first anchor to the membrane, were not suited for this task. We hypothesized this is likely because cell membranes can wrap around the dihedral components’ identical two sides, each displaying an equal number of binding sites, thereby blocking assembly. We therefore devised cyclic pseudo-dihedral versions of the array components, which are partly dihedral to allow proper formation of a 2D array, but overall cyclic to allow directional anchoring.
Because the components are individually stable in ambient conditions and peptide fusion provides a rapid and simple way to diversify the components’ functionality prior to array formation (and thereby the system functionality post-array formation), we fused to either or both components optical labels as well as ligands to specifically interact with cell surface receptors. A partial list includes: sfGPF to GBP, a GFP binding peptide, F domain to Tie2, EGFR-binder5 to EGFR, etc. We exploit the system’s multi-parameter controllability to demonstrate formation of biologically-active structures on mammalian cell membranes to tunable dimensions, showcasing ordered receptor clustering and tunable endocytosis properties.
The system’s functional modularity, temporal controllability, and targeted location assembly demonstrated here will enable new methods for characterization of structural-functional relations in living systems. This work marries protein engineering and cell biology into a brand new field that would allow probing and elucidating fundamental biological questions.
1. Ben-Sasson, A. J. et al. Design of Biologically Active Binary Protein 2D Materials. bioRxiv 2020.09.19.304253 (2020) doi:10.1101/2020.09.19.304253.
2. Sleytr, U. B., Schuster, B., Egelseer, E.-M. & Pum, D. S-layers: principles and applications. FEMS Microbiol Rev 38, 823–864 (2014).
3. Baneyx, F. & Matthaei, J. F. Self-assembled two-dimensional protein arrays in bionanotechnology: from S-layers to designed lattices. Current Opinion in Biotechnology 28, 39–45 (2014).
4. Vantomme, G. & Meijer, E. W. The construction of supramolecular systems. Science 363, 1396–1397 (2019).
5. Pedersen, M. W. et al. Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy. Cancer Res 70, 588–597 (2010).