Chaotrope-Based Approach for Rapid In Vitro Assembly and Loading of Bacterial Microcompartment Shells

Range, Kyleigh L. [Michigan State University, East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0009000943337539); Chiang, Timothy K. [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0009000058119221); Pramanik, Arinita [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)]; Landa, Joel F. [Michigan State University, East Lansing, MI (United States)] (ORCID:0000000318826698); Snyder, Samuel N. [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000225714048); Zuo, Xiaobing [Argonne National Laboratory (ANL), Argonne, IL (United States)]; Tiede, David M. [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000227844954); Utschig, Lisa M. [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000320955392); Hegg, Eric L. [Michigan State University, East Lansing, MI (United States)] (ORCID:0000000320555495); Sutter, Markus [Michigan State University, East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000162904820); Kerfeld, Cheryl A. [Michigan State University, East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000299778482)

doi:10.1021/acsnano.4c15538

DOE OSTI · 2533606

Chaotrope-Based Approach for Rapid In Vitro Assembly and Loading of Bacterial Microcompartment Shells

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Abstract

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here, we describe the development of an efficient and rapid method for the in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY 59 BASIC BIOLOGICAL SCIENCES bacterial microcompartments biotic and abiotic cargo encapsulation catalysis confinement encapsulation genetics in vitro peptides and proteins self-assembly size exclusion chromotography urea

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Range, Kyleigh L. [Michigan State University, East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0009000943337539), Chiang, Timothy K. [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0009000058119221), Pramanik, Arinita [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)], Landa, Joel F. [Michigan State University, East Lansing, MI (United States)] (ORCID:0000000318826698), Snyder, Samuel N. [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000225714048), Zuo, Xiaobing [Argonne National Laboratory (ANL), Argonne, IL (United States)], Tiede, David M. [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000227844954), Utschig, Lisa M. [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000320955392), Hegg, Eric L. [Michigan State University, East Lansing, MI (United States)] (ORCID:0000000320555495), Sutter, Markus [Michigan State University, East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000162904820), Kerfeld, Cheryl A. [Michigan State University, East Lansing, MI (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:0000000299778482). 2025-03-20. Chaotrope-Based Approach for Rapid In Vitro Assembly and Loading of Bacterial Microcompartment Shells. https://doi.org/10.1021/acsnano.4c15538

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