Engineering the entropy-driven free-energy landscape of a dynamic nanoporous protein assembly

ABSTRACT De novo design and construction of stimuli-responsive protein assemblies that predictably switch between discrete conformational states remains an essential but highly challenging

goal in biomolecular design. We previously reported synthetic, two-dimensional protein lattices self-assembled via disulfide bonding interactions, which endows them with a unique capacity to

undergo coherent conformational changes without losing crystalline order. Here, we carried out all-atom molecular dynamics simulations to map the free-energy landscape of these lattices,

validated this landscape through extensive structural characterization by electron microscopy and established that it is predominantly governed by solvent reorganization entropy. Subsequent

redesign of the protein surface with conditionally repulsive electrostatic interactions enabled us to predictably perturb the free-energy landscape and obtain a new protein lattice whose

conformational dynamics can be chemically and mechanically toggled between three different states with varying porosities and molecular densities. Access through your institution Buy or

subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get

Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn more Subscribe to this journal Receive 12 print issues and online access $259.00 per year only

$21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout

ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ASSEMBLY OF A PATCHY PROTEIN

INTO VARIABLE 2D LATTICES VIA TUNABLE MULTISCALE INTERACTIONS Article Open access 28 July 2020 HIERARCHICAL DESIGN OF PSEUDOSYMMETRIC PROTEIN NANOCAGES Article Open access 18 December 2024

COMPLETE AND COOPERATIVE IN VITRO ASSEMBLY OF COMPUTATIONALLY DESIGNED SELF-ASSEMBLING PROTEIN NANOMATERIALS Article Open access 09 February 2021 REFERENCES * Marsh, J. A. & Teichmann,

S. A. Structure, dynamics, assembly, and evolution of protein complexes. _Annu. Rev. Biochem._ 84, 551–575 (2015). Article  CAS  PubMed  Google Scholar  * Salgado, E. N., Radford, R. J.

& Tezcan, F. A. Metal-directed protein self-assembly. _Acc. Chem. Res._ 43, 661–672 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Song, W. J. & Tezcan, F. A. A

designed supramolecular protein assembly with in vivo enzymatic activity. _Science_ 346, 1525–1528 (2014). Article  CAS  PubMed  Google Scholar  * Yeates, T. O. Geometric principles for

designing highly symmetric self-assembling protein nanomaterials. _Annu. Rev. Biophys._ 46, 23–42 (2017). Article  CAS  PubMed  Google Scholar  * Bale, J. B. et al. Accurate design of

megadalton-scale two-component icosahedral protein complexes. _Science_ 353, 389–394 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Koga, N. et al. Principles for designing

ideal protein structures. _Nature_ 491, 222–227 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Norn, C. H. & André, I. Computational design of protein self-assembly.

_Curr. Opin. Struct. Biol._ 39, 39–45 (2016). Article  CAS  PubMed  Google Scholar  * Ringler, P. & Schulz, G. E. Self-assembly of proteins into designed networks. _Science_ 302, 106–109

(2003). Article  CAS  PubMed  Google Scholar  * Sinclair, J. C., Davies, K. M., Venien-Bryan, C. & Noble, M. E. M. Generation of protein lattices by fusing proteins with matching

rotational symmetry. _Nat. Nanotechnol._ 6, 558–562 (2011). Article  CAS  PubMed  Google Scholar  * Levy, Y. & Onuchic, J. N. Water mediation in protein folding and molecular

recognition. _Annu. Rev. Biophys. Biomol. Struct._ 35, 389–415 (2006). Article  CAS  PubMed  Google Scholar  * Young, T., Abel, R., Kim, B., Berne, B. J. & Friesner, R. A. Motifs for

molecular recognition exploiting hydrophobic enclosure in protein–ligand binding. _Proc. Natl Acad. Sci. USA_ 104, 808–813 (2007). Article  CAS  PubMed  Google Scholar  * Hedstrom, L. Serine

protease mechanism and specificity. _Chem. Rev._ 102, 4501–4524 (2002). Article  CAS  PubMed  Google Scholar  * Davey, J. A., Damry, A. M., Goto, N. K. & Chica, R. A. Rational design of

proteins that exchange on functional timescales. _Nat. Chem. Biol._ 13, 1280–1285 (2017). Article  CAS  PubMed  Google Scholar  * Joh, N. H. et al. De novo design of a transmembrane

Zn2+-transporting four-helix bundle. _Science_ 346, 1520–1524 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cerasoli, E., Sharpe, B. K. & Woolfson, D. N. ZiCo: a

peptide designed to switch folded state upon binding zinc. _J. Am. Chem. Soc._ 127, 15008–15009 (2005). Article  CAS  PubMed  Google Scholar  * Ambroggio, X. I. & Kuhlman, B.

Computational design of a single amino acid sequence that can switch between two distinct protein folds. _J. Am. Chem. Soc._ 128, 1154–1161 (2006). Article  CAS  PubMed  Google Scholar  *

Brodin, J. D. et al. Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. _Nat. Chem._ 4, 375–382 (2012). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Sontz, P. A., Bailey, J. B., Ahn, S. & Tezcan, F. A. A metal organic framework with spherical protein nodes: rational chemical design of 3D protein crystals.

_J. Am. Chem. Soc._ 137, 11598–11601 (2015). Article  CAS  PubMed  Google Scholar  * Bailey, J. B., Subramanian, R. H., Churchfield, L. A. & Tezcan, F. A. Metal-directed design of

supramolecular protein assemblies. _Methods Enzymol._ 580, 223–250 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Suzuki, Y. et al. Self-assembly of coherently dynamic,

auxetic, two-dimensional protein crystals. _Nature_ 533, 369–373 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Grima, J. N. & Evans, K. E. Auxetic behavior from

rotating squares. _J. Mater. Sci. Lett._ 19, 1563–1565 (2000). Article  CAS  Google Scholar  * Yang, W., Li, Z.-M., Shi, W., Xie, B.-H. & Yang, M.-B. Review on auxetic materials. _J.

Mater. Sci._ 39, 3269–3279 (2004). Article  CAS  Google Scholar  * Evans, K. E. & Alderson, A. Auxetic materials: functional materials and structures from lateral thinking! _Adv. Mater._

12, 617–628 (2000). Article  CAS  Google Scholar  * Zhu, F. & Hummer, G. Convergence and error estimation in free energy calculations using the weighted histogram analysis method. _J.

Comput. Chem._ 33, 453–465 (2012). Article  CAS  PubMed  Google Scholar  * Kumar, S., Rosenberg, J. M., Bouzida, D., Swendsen, R. H. & Kollman, P. A. The weighted histogram analysis

method for free-energy calculations on biomolecules. I. The method. _J. Comput. Chem._ 13, 1011–1021 (1992). Article  CAS  Google Scholar  * De Yoreo, J. J. & Vekilov, P. G. Principles

of crystal nucleation and growth. _Rev. Mineral. Geochem._ 54, 57–93 (2003). Article  CAS  Google Scholar  * Derewenda, Z. S. & Vekilov, P. G. Entropy and surface engineering in protein

crystallization. _Acta Crystallogr. D_ 62, 116–124 (2006). Article  CAS  PubMed  Google Scholar  * Nguyen, C. N., Young, T. K. & Gilson, M. K. Grid inhomogeneous solvation theory:

hydration structure and thermodynamics of the miniature receptor cucurbit[7]uril. _J. Chem. Phys._ 137, 044101 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Huggins, D. J.

Comparing distance metrics for rotation using the k-nearest neighbors algorithm for entropy estimation. _J. Comput. Chem._ 35, 377–385 (2014). Article  CAS  PubMed  Google Scholar  *

Giovambattista, N., Rossky, P. J. & Debenedetti, P. G. Effect of temperature on the structure and phase behavior of water confined by hydrophobic, hydrophilic, and heterogeneous

surfaces. _J. Phys. Chem. B_ 113, 13723–13734 (2009). Article  CAS  PubMed  Google Scholar  * Tarek, M. & Tobias, D. J. The dynamics of protein hydration water: a quantitative comparison

of molecular dynamics simulations and neutron-scattering experiments. _Biophys. J._ 79, 3244–3257 (2000). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ebbinghaus, S. et al. An

extended dynamical hydration shell around proteins. _Proc. Natl Acad. Sci. USA_ 104, 20749–20752 (2007). Article  PubMed  Google Scholar  * Heyden, M. et al. Dissecting the THz spectrum of

liquid water from first principles via correlations in time and space. _Proc. Natl Acad. Sci. USA_ 107, 12068–12073 (2010). Article  PubMed  Google Scholar  * Dunitz, J. D. The entropic cost

of bound water in crystals and biomolecules. _Science_ 264, 670 (1994). Article  CAS  PubMed  Google Scholar  * Yuan, Y., Tam, M. F., Simplaceanu, V. & Ho, C. New look at hemoglobin

allostery. _Chem. Rev._ 115, 1702–1724 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Joshi, R. K. et al. Precise and ultrafast molecular sieving through graphene oxide

membranes. _Science_ 343, 752–754 (2014). Article  CAS  PubMed  Google Scholar  * Tahara, K., Nakatani, K., Iritani, K., De Feyter, S. & Tobe, Y. Periodic functionalization of

surface-confined pores in a two-dimensional porous network using a tailored molecular building block. _ACS Nano_ 10, 2113–2120 (2016). Article  CAS  PubMed  Google Scholar  * Huber, C. et

al. Heterotetramers formed by an S-layer–streptavidin fusion protein and core-streptavidin as a nanoarrayed template for biochip development. _Small_ 2, 142–150 (2006). Article  CAS  PubMed

  Google Scholar  * Li, Y. et al. MoS2 nanoparticles grown on graphene: an advanced catalyst for the hydrogen evolution reaction. _J. Am. Chem. Soc._ 133, 7296–7299 (2011). Article  CAS 

PubMed  Google Scholar  * Mader, C., Küpcü, S., Sleytr, U. B. & Sára, M. S-layer-coated liposomes as a versatile system for entrapping and binding target molecules. _Biochim. Biophys.

Acta Biomembr._ 1463, 142–150 (2000). Article  CAS  Google Scholar  * Butler, S. Z. et al. Progress, challenges, and opportunities in two-dimensional materials beyond graphene. _ACS Nano_ 7,

2898–2926 (2013). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M. Gilson, T. Kurtzman and S. Ramsey for helpful discussions regarding GIST, R.

Subramanian for assistance with the generation of projection maps from computational models, and T. Baker for use of the electron microscopy facilities. This work was primarily supported by

the US Department of Energy (Division of Materials Sciences, Office of Basic Energy Sciences; Award DE-SC0003844 to F.A.T.). F.P. was supported by the National Science Foundation through

grant CHE-1453204 (computation). R.A. was supported in part by the University of California, San Diego NIH Molecular Biophysics Training Grant (T32-GM08326). All computer simulations were

performed on the Extreme Science and Engineering Discovery Environment, which is supported by the National Science Foundation through grant ACI-1053575. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA Robert Alberstein, Yuta Suzuki, Francesco Paesani & F. Akif Tezcan *

Materials Science and Engineering, University of California, San Diego, La Jolla, CA, USA Francesco Paesani & F. Akif Tezcan Authors * Robert Alberstein View author publications You can

also search for this author inPubMed Google Scholar * Yuta Suzuki View author publications You can also search for this author inPubMed Google Scholar * Francesco Paesani View author

publications You can also search for this author inPubMed Google Scholar * F. Akif Tezcan View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

R.A. co-initiated the project, designed and performed all of the experiments, simulations and data analysis, and co-wrote the paper. Y.S. performed the TEM data collection and analysis. F.P.

guided the simulation design and computational data analysis, and co-wrote the paper. F.A.T. initiated the project, guided the experiment design and data analysis, and co-wrote the paper.

CORRESPONDING AUTHORS Correspondence to Francesco Paesani or F. Akif Tezcan. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION

Methods, Supplementary Figures 1–11, Supplementary Table 1 and Supplementary References RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Alberstein, R.,

Suzuki, Y., Paesani, F. _et al._ Engineering the entropy-driven free-energy landscape of a dynamic nanoporous protein assembly. _Nature Chem_ 10, 732–739 (2018).

https://doi.org/10.1038/s41557-018-0053-4 Download citation * Received: 04 December 2017 * Accepted: 29 March 2018 * Published: 30 April 2018 * Issue Date: July 2018 * DOI:

https://doi.org/10.1038/s41557-018-0053-4 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative