Elastin-like polypeptides (ELPs) are biocompatible polymers exhibiting lower critical solution temperature (LCST) behavior, making them valuable in various applications, including drug delivery and tissue engineering. This study addresses the atomistic-level understanding of ELP self-assembly, focusing on their internal structural dynamics. Conventional proton-detected nuclear magnetic resonance (NMR) spectroscopy faces limitations in studying ELP aggregates due to accelerated proton exchange processes, which cause significant resonance broadening. Herein, we show how to overcome this hurdle by using carbon-13-detected NMR. This method mitigates issues related to amide proton exchange, allowing for a residue-resolved view of the internal configuration of ELP aggregates. With this method, we record residue-resolved 15N relaxation rates, revealing three features. (i) Proline residues within the PGXGV pentapeptide repeats (X being any amino acid except proline) of ELP become motional restricted upon aggregation, indicating their role as interchain contacts. (ii) Pentapeptides with alanine guest residue X display particularly significantly reduced motional freedom upon aggregation. (iii) Even within large ELP aggregates, fast internal dynamics characterize the peptide chains in a way that is reminiscent of condensed liquid phases. The presented study is the first proof of concept that 13C-direct detection is a viable tool to delineate the internal structural dynamics of condensed ELP phases by NMR. It might, thus, help to foster new investigations of their aggregation mechanisms.

Central European Institute of Technology Masaryk University Kamenice 5 625 00 Brno Czech Republic

Institute of Biological Chemistry Faculty of Chemistry University of Vienna Währinger Str 38 1090 Vienna Austria

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Vienna Doctoral School in Chemistry University of Vienna Währinger Str 42 1090 Vienna Austria

See more in PubMed

Hyun J.; Lee W. K.; Nath N.; Chilkoti A.; Zauscher S. Capture and release of proteins on the nanoscale by stimuli-responsive elastin-like polypeptide ″switches″. J. Am. Chem. Soc. 2004, 126, 7330–7335. 10.1021/ja049721e. PubMed DOI

Osborne J. L.; Farmer R.; Woodhouse K. A. Self-assembled elastin-like polypeptide particles. Acta Biomat. 2008, 4, 49–57. 10.1016/j.actbio.2007.07.007. PubMed DOI

Wu W. Y.; Fong B. A.; Gilles A. G.; Wood D. W. Recombinant protein purification by self-cleaving elastin-like polypeptide fusion tag. Curr. Protoc Protein Sci. 2009, 26, na.10.1002/0471140864.ps2604s58. PubMed DOI

Hassouneh W.; Christensen T.; Chilkoti A. Elastin-like polypeptides as a purification tag for recombinant proteins. Curr. Protoc. Protein Sci. 2010, 61, na.10.1002/0471140864.ps0611s61. PubMed DOI PMC

Hassouneh W.; Zhulina E. B.; Chilkoti A.; Rubinstein M. Elastin-like Polypeptide Diblock Copolymers Self-Assemble into Weak Micelles. Macromolecules 2015, 48, 4183–4195. 10.1021/acs.macromol.5b00431. PubMed DOI PMC

Reichheld S. E.; Muiznieks L. D.; Keeley F. W.; Sharpe S. Direct observation of structure and dynamics during phase separation of an elastomeric protein. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E4408–E4415. 10.1073/pnas.1701877114. PubMed DOI PMC

Rubinstein M.; Colby R. H.. Polymer Physics; Oxfod University Press: New York, 2003, Vol. 23.

Betre H.; Ong S. O.; Guilak F.; Chilkoti A.; Fermor B.; Setton L. A. Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide. Biomaterials 2006, 27, 91–99. 10.1016/j.biomaterials.2005.05.071. PubMed DOI

Kurzbach D.; Hassouneh W.; McDaniel J. R.; Jaumann E. A.; Chilkoti A.; Hinderberger D. Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers. J. Am. Chem. Soc. 2013, 135, 11299–11308. 10.1021/ja4047872. PubMed DOI PMC

Han W.; MacEwan S. R.; Chilkoti A.; Lopez G. P. Bio-inspired synthesis of hybrid silica nanoparticles templated from elastin-like polypeptide micelles. Nanoscale 2015, 7, 12038–12044. 10.1039/C5NR01407G. PubMed DOI PMC

Zhang Y.-N.; Avery R. K.; Vallmajo-Martin Q.; Assmann A.; Vegh A.; Memic A.; Olsen B. D.; Annabi N.; Khademhosseini A. A Highly Elastic and Rapidly Crosslinkable Elastin-Like Polypeptide-Based Hydrogel for Biomedical Applications. Adv. Funct Mater. 2015, 25, 4814–4826. 10.1002/adfm.201501489. PubMed DOI PMC

Hu J.; Wang G.; Liu X.; Gao W. Enhancing Pharmacokinetics, Tumor Accumulation, and Antitumor Efficacy by Elastin-Like Polypeptide Fusion of Interferon Alpha. Adv. Mater. 2015, 27, 7320–7324. 10.1002/adma.201503440. PubMed DOI

Ott W.; Jobst M. A.; Bauer M. S.; Durner E.; Milles L. F.; Nash M. A.; Gaub H. E. Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy. ACS Nano 2017, 11, 6346–6354. 10.1021/acsnano.7b02694. PubMed DOI

Weißheit S.; Kahse M.; Kämpf K.; Tietze A.; Vogel M.; Winter R.; Thiele C. M. Elastin-like Peptide in Confinement: FT-IR and NMR T1 Relaxation Data. Zeitschrift für Naturforschung 2017, 232, 7–8.

Gurumurthy B.; Tucci M. A.; Fan L. W.; Benghuzzi H. A.; Pal P.; Bidwell G. L.; Salazar Marocho S. M.; Cason Z.; Gordy D.; Janorkar A. V. Collagen-Elastin-Like Polypeptide-Bioglass Scaffolds for Guided Bone Regeneration. Adv. Healthc Mater. 2020, 9, e190138510.1002/adhm.201901385. PubMed DOI

Nelson D. W.; Gilbert R. J. Extracellular Matrix-Mimetic Hydrogels for Treating Neural Tissue Injury: A Focus on Fibrin, Hyaluronic Acid, and Elastin-Like Polypeptide Hydrogels. Adv. Healthc Mater. 2021, 10, e210132910.1002/adhm.202101329. PubMed DOI PMC

Shi X.; Chen D.; Liu G.; Zhang H.; Wang X.; Wu Z.; Wu Y.; Yu F.; Xu Q. Application of Elastin-like Polypeptide in Tumor Therapy. Cancers (Basel) 2022, 14, 3683.10.3390/cancers14153683. PubMed DOI PMC

Milligan J. J.; Saha S.; Jenkins I. C.; Chilkoti A. Genetically encoded elastin-like polypeptide nanoparticles for drug delivery. Curr. Opin Biotechnol 2022, 74, 146–153. 10.1016/j.copbio.2021.11.006. PubMed DOI

Li Y.; Dautel D. R.; Gray M. A.; McKenna M. E.; Champion J. A. Rational design of elastin-like polypeptide fusion proteins to tune self-assembly and properties of protein vesicles. J. Mater. Chem. B 2023, 11, 6443–6452. 10.1039/D3TB00200D. PubMed DOI

van Strien J.; Escalona-Rayo O.; Jiskoot W.; Slutter B.; Kros A. Elastin-like polypeptide-based micelles as a promising platform in nanomedicine. J. Controlled Release 2023, 353, 713–726. 10.1016/j.jconrel.2022.12.033. PubMed DOI

Lopez Barreiro D.; Minten I. J.; Thies J. C.; Sagt C. M. J. Structure-Property Relationships of Elastin-like Polypeptides: A Review of Experimental and Computational Studies. ACS Biomater Sci. Eng. 2023, 9, 3796–3809. 10.1021/acsbiomaterials.1c00145. PubMed DOI

Hassouneh W.; Christensen T.; Chilkoti A. Elastin-like polypeptides as a purification tag for recombinant proteins. Curr. Protoc Protein Sci. 2010, 61, na.10.1002/0471140864.ps0611s61. PubMed DOI PMC

Urry D. W. Elastic molecular machines in metabolism and soft-tissue restoration. Trends Biotechnol 1999, 17, 249–257. 10.1016/S0167-7799(99)01306-2. PubMed DOI

Kim J.-Y.; O’Malley S.; Mulchandani A.; Chen W. Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay. Anal. Chem. 2005, 77, 2318–2322. 10.1021/ac0484326. PubMed DOI

Valiaev A.; Abu-Lail N. I.; Lim D. W.; Chilkoti A.; Zauscher S. Microcantilever sensing and actuation with end-grafted stimulus-responsive elastin-like polypeptides. Langmuir 2007, 23, 339–344. 10.1021/la0616698. PubMed DOI

Li N. K.; Quiroz F. G.; Hall C. K.; Chilkoti A.; Yingling Y. G. Molecular description of the LCST behavior of an elastin-like polypeptide. Biomacromolecules 2014, 15, 3522–3530. 10.1021/bm500658w. PubMed DOI

Reichheld S. E.; Muiznieks L. D.; Keeley F. W.; Sharpe S. Direct observation of structure and dynamics during phase separation of an elastomeric protein. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E4408–E4415. 10.1073/pnas.1701877114. PubMed DOI PMC

Urry D. W.; Trapane T. L.; Iqbal M.; Venkatachalam C. M.; Prasad K. U. Carbon-13 NMR relaxation studies demonstrate an inverse temperature transition in the elastin polypentapeptide. Biochemistry 1985, 24, 5182–5189. 10.1021/bi00340a034. PubMed DOI

Li B.; Alonso D. O.; Bennion B. J.; Daggett V. Hydrophobic hydration is an important source of elasticity in elastin-based biopolymers. J. Am. Chem. Soc. 2001, 123, 11991–11998. 10.1021/ja010363e. PubMed DOI

Yao X.; Hong M. Structure distribution in an elastin-mimetic peptide (PGVGV) 3 investigated by solid-state NMR. J. Am. Chem. Soc. 2004, 126, 4199–4210. 10.1021/ja036686n. PubMed DOI

Glaves R.; Baer M.; Schreiner E.; Stoll R.; Marx D. Conformational dynamics of minimal elastin-like polypeptides: the role of proline revealed by molecular dynamics and nuclear magnetic resonance. ChemPhysChem 2008, 9, 2759–2765. 10.1002/cphc.200800474. PubMed DOI

Sun C.; Mitchell O.; Huang J.; Boutis G. S. NMR studies of localized water and protein backbone dynamics in mechanically strained elastin. J. Phys. Chem. B 2011, 115, 13935–13942. 10.1021/jp207607r. PubMed DOI PMC

Weißheit S.; Kahse M.; Kämpf K.; Tietze A.; Vogel M.; Winter R.; Thiele C. M. Elastin-like Peptide in Confinement: FT-IR and NMR T 1 Relaxation Data. Z. Phys. Chem. 2018, 232 (2018), 1239–1261. 10.1515/zpch-2017-1047. DOI

Rule G.; Hitchens K.. Fundamentals of Protein NMR Spectroscopy 1st ed.; Springer: Dordrecht, 2006.

Urry D. W.; Parker T. M. Mechanics of elastin: molecular mechanism of biological elasticity and its relationship to contraction. J. Muscle Res. Cell Motil 2002, 23, 543–559. 10.1023/A:1023422925910. PubMed DOI

Spěváček J. NMR investigations of phase transition in aqueous polymer solutions and gels. Curr. Opin. Colloid Interface Sci. 2009, 14, 184–191. 10.1016/j.cocis.2008.10.003. DOI

Heatley F. Nuclear magnetic relaxation of synthetic polymers in dilute solution. Prog. Nucl. Mag Res. Sp 1979, 13, 47–85. 10.1016/0079-6565(79)80013-8. DOI

Bovey F. A.; Jelinski L. The observation of chain motion in macromolecules by carbon-13 and deuterium nuclear magnetic resonance spectroscopy. J. Phys. Chem. 1985, 89, 571–583. 10.1021/j100250a006. DOI

Schmidt-Rohr K.; Spiess H. W.. Multidimensional solid-state NMR and polymers; Elsevier, 2012.

Urry D. W.; Harris R. D.; Long M. M. Compounding of elastin polypentapeptide to collagen analogue: a potential elastomeric prosthetic material. Biomater Med. Devices Artif Organs 1981, 9, 181–194. 10.3109/10731198109118999. PubMed DOI

Urry D. W.; Luan C. H.; Peng S. Q. Molecular biophysics of elastin structure, function and pathology. Ciba Foundation Symposium 192, The Molecular Biology and Pathology of Elastic Tissues 2007, 192, 4–30. 10.1002/9780470514771.ch2. PubMed DOI

Delaglio F.; Grzesiek S.; Vuister G. W.; Zhu G.; Pfeifer J.; Bax A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol NMR 1995, 6, 277–293. 10.1007/BF00197809. PubMed DOI

Lee W.; Tonelli M.; Markley J. L. NMRFAM-SPARKY: Enhanced software for biomolecular NMR spectroscopy. Bioinformatics 2015, 31, 1325–1327. 10.1093/bioinformatics/btu830. PubMed DOI PMC

Castanar L.; Poggetto G. D.; Colbourne A. A.; Morris G. A.; Nilsson M. The GNAT: A new tool for processing NMR data. Magn. Reson. Chem. 2018, 56, 546–558. 10.1002/mrc.4717. PubMed DOI PMC

Tanford C.; Huggins M. L. Physical Chemistry of Macromolecules. J. Electrochem. Soc. 1962, 109, 98C.10.1149/1.2425390. DOI

Einstein A. Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen. Annalen der Physik 1905, 322, 549–560. 10.1002/andp.19053220806. DOI

Stokes G. G. On the Effect of the Internal Friction of Fluids on the Motion of Pendulums. Transactions of the Chambridge Philosophical Society 1850, 11, 8–96.

Vranken W. F.; Boucher W.; Stevens T. J.; Fogh R. H.; Pajon A.; Llinas M.; Ulrich E. L.; Markley J. L.; Ionides J.; Laue E. D. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 2005, 59, 687–696. 10.1002/prot.20449. PubMed DOI

Lopez J.; Ahuja P.; Landrieu I.; Cantrelle F. X.; Huvent I.; Lippens G. H/D exchange of a 15N labelled Tau fragment as measured by a simple Relax-EXSY experiment. J. Magn. Reson. 2014, 249, 32–37. 10.1016/j.jmr.2014.10.008. PubMed DOI

Kurzbach D.; Hassouneh W.; McDaniel J. R.; Jaumann E. A.; Chilkoti A.; Hinderberger D. Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers. J. Am. Chem. Soc. 2013, 135, 11299–11308. 10.1021/ja4047872. PubMed DOI PMC

Abramov G.; Velyvis A.; Rennella E.; Wong L. E.; Kay L. E. A methyl-TROSY approach for NMR studies of high-molecular-weight DNA with application to the nucleosome core particle. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 12836–12846. 10.1073/pnas.2004317117. PubMed DOI PMC

Rossler P.; Mathieu D.; Gossert A. D. Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling. Angew. Chem., Int. Ed. Engl. 2020, 59, 19329–19337. 10.1002/anie.202007715. PubMed DOI PMC

Tugarinov V.; Ceccon A.; Clore G. M. NMR methods for exploring ’dark’ states in ligand binding and protein-protein interactions. Prog. Nucl. Magn. Reson. Spectrosc. 2022, 128, 1–24. 10.1016/j.pnmrs.2021.10.001. PubMed DOI PMC

Bolik-Coulon N.; Sever A. I. M.; Harkness R. W.; Aramini J. M.; Toyama Y.; Hansen D. F.; Kay L. E. Less is more: A simple methyl-TROSY based pulse scheme offers improved sensitivity in applications to high molecular weight complexes. J. Magn. Reson. 2023, 346, 107326.10.1016/j.jmr.2022.107326. PubMed DOI

Unarta I. C.; Cao S.; Goonetilleke E. C.; Niu J.; Gellman S. H.; Huang X. Submillisecond Atomistic Molecular Dynamics Simulations Reveal Hydrogen Bond-Driven Diffusion of a Guest Peptide in Protein-RNA Condensate. J. Phys. Chem. B 2024, 128, 2347–2359. 10.1021/acs.jpcb.3c08126. PubMed DOI PMC

Rauscher S.; Pomes R. The liquid structure of elastin. Elife 2017, 6, e26526.10.7554/eLife.26526. PubMed DOI PMC

Bai Y.; Milne J. S.; Mayne L.; Englander S. W. Primary structure effects on peptide group hydrogen exchange. Proteins 1993, 17, 75–86. 10.1002/prot.340170110. PubMed DOI PMC

Felli I. C.; Brutscher B. Recent advances in solution NMR: fast methods and heteronuclear direct detection. ChemPhysChem 2009, 10, 1356–1368. 10.1002/cphc.200900133. PubMed DOI

Madl T.; Felli I. C.; Bertini I.; Sattler M. Structural analysis of protein interfaces from 13C direct-detected paramagnetic relaxation enhancements. J. Am. Chem. Soc. 2010, 132, 7285–7287. 10.1021/ja1014508. PubMed DOI

Bermel W.; Bertini I.; Chill J.; Felli I. C.; Haba N.; Kumar M. V V.; Pierattelli R. Exclusively heteronuclear (13) C-detected amino-acid-selective NMR experiments for the study of intrinsically disordered proteins (IDPs). Chembiochem 2012, 13, 2425–2432. 10.1002/cbic.201200447. PubMed DOI

Felli I. C.; Pierattelli R. Novel methods based on (13)C detection to study intrinsically disordered proteins. J. Magn. Reson. 2014, 241, 115–125. 10.1016/j.jmr.2013.10.020. PubMed DOI

Felli I. C.; Pierattelli R. (13)C Direct Detected NMR for Challenging Systems. Chem. Rev. 2022, 122, 9468–9496. 10.1021/acs.chemrev.1c00871. PubMed DOI PMC

Schiavina M.; Bracaglia L.; Rodella M. A.; Kümmerle R.; Konrat R.; Felli I. C.; Pierattelli R. Optimal (13)C NMR investigation of intrinsically disordered proteins at 1.2 GHz. Nat. Protoc 2024, 19, 406.10.1038/s41596-023-00921-9. PubMed DOI

Murrali M. G.; Piai A.; Bermel W.; Felli I. C.; Pierattelli R. Proline Fingerprint in Intrinsically Disordered Proteins. Chembiochem 2018, 19, 1625–1629. 10.1002/cbic.201800172. PubMed DOI

Kowalewski J.; Nordenskiöld L.; Benetis N.; Westlund P.-O. Theory of nuclear spin relaxation in paramagnetic systems in solution. Prog. Nucl. Mag Res. Sp 1985, 17, 141–185. 10.1016/0079-6565(85)80007-8. DOI

Schiavina M.; Konrat R.; Ceccolini I.; Mateos B.; Konrat R.; Felli I. C.; Pierattelli R. Studies of proline conformational dynamics in IDPs by (13)C-detected cross-correlated NMR relaxation. J. Magn. Reson. 2023, 354, 10753910.1016/j.jmr.2023.107539. PubMed DOI

Carper W. R.; Keller C. E. Direct determination of NMR correlation times from spin– lattice and spin– spin relaxation times. J. Phys. Chem. A 1997, 101 (1997), 3246–3250. 10.1021/jp963338h. DOI

Toyama Y.; Rangadurai A. K.; Forman-Kay J. D.; Kay L. E. Mapping the per-residue surface electrostatic potential of CAPRIN1 along its phase-separation trajectory. Proc. Natl. Acad. Sci. U. S. A. 2022, 119, e221049211910.1073/pnas.2210492119. PubMed DOI PMC

Guseva S.; Schnapka V.; Adamski W.; Maurin D.; Ruigrok R. W. H.; Salvi N.; Blackledge M. Liquid-Liquid Phase Separation Modifies the Dynamic Properties of Intrinsically Disordered Proteins. J. Am. Chem. Soc. 2023, 145, 10548–10563. 10.1021/jacs.2c13647. PubMed DOI PMC

Evans J. S. Liquid-like” biomineralization protein assemblies: a key to the regulation of non-classical nucleation. CrystEngComm 2013, 15, 8388.10.1039/c3ce40803e. DOI

Polyansky A. A.; Gallego L. D.; Efremov R. G.; Kohler A.; Zagrovic B. Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales. Elife 2023, 12, e80038.10.7554/eLife.80038. PubMed DOI PMC

Guo Y.; Liu S.; Jing D.; Liu N.; Luo X. The construction of elastin-like polypeptides and their applications in drug delivery system and tissue repair. J. Nanobiotechnology 2023, 21, 418.10.1186/s12951-023-02184-8. PubMed DOI PMC

Panchal S. C.; Bhavesh N. S.; Hosur R. V. Improved 3D triple resonance experiments, HNN and HN(C)N, for HNand 15N sequential correlations in (13C, 15N) labeled proteins: Application to unfolded proteins. J. of Biomol NMR 2001, 20, 135–147. 10.1023/A:1011239023422. PubMed DOI