Dataset Information

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

ABSTRACT: Engineering a protein variant with a desired role relies on deep knowledge of the relationship between a protein's native structure and function. Using our structural understanding of a regulatory subdomain found in a family of DNA helicases, we engineered novel helicases for which the subdomain orientation is designed to switch between unwinding-inactive and -active conformations upon trans-cis isomerization of an azobenzene-based crosslinker. This on-demand light-based conformational control directly alters helicase activity as demonstrated by both bulk phase experiments and single-molecule optical tweezers analysis of one of the engineered helicases. The "opto-helicase" may be useful in future applications that require spatiotemporal control of DNA hybridization states.

SUBMITTER: Bobrovnikov D

PROVIDER: S-EPMC10557133 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Bobrovnikov Dmitriy D Makurath Monika A MA Wolfe Clara H CH Chemla Yann R YR Ha Taekjip T

Journal of the American Chemical Society 20230922 39

Engineering a protein variant with a desired role relies on deep knowledge of the relationship between a protein's native structure and function. Using our structural understanding of a regulatory subdomain found in a family of DNA helicases, we engineered novel helicases for which the subdomain orientation is designed to switch between unwinding-inactive and -active conformations upon <i>trans-cis</i> isomerization of an azobenzene-based crosslinker. This on-demand light-based conformational co ...[more]

PMID: 37739407

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Project description:We previously described the photoactivated depot or PAD approach that allows for the light control of therapeutic protein release. This approach relies on the ability to use light to change a protein's solubility. Traditionally this was accomplished by linking the protein to an insoluble but injectable polymer via a light cleaved linker. This allows the injected material to remain at the site of injection, until transcutaneous irradiation breaks the link between polymer and protein, permitting the protein to be absorbed. However, there are multiple problems associated with polymer based approaches: The polymer makes up a majority of the material, making it inefficient. In addition, after protein release, the polymer has to be cleared from the body, a significant design challenge. In this work, we create materials that form photoactivated depots of insulin without the need for polymers, by linking photolysis to an isoelectric point shift, which itself is linked to a solubility shift. Specifically, we linked basic groups to insulin via a light cleaved linker. These shift the normal pI of insulin from 5.4 to approximately 7. The result of this incorporation are materials that are completely soluble in mildly acidic solutions but precipitate upon injection into a pH 7 environment, i.e., the skin. We successfully synthesized four such modified insulins, demonstrating that their pI values were shifted in the expected manner. We then analyzed one of them, P2-insulin, in detail, demonstrating that it behaves as designed: It is soluble in a formulation pH of 4, but precipitates at pH 7.2, its approximate pI value. Upon irradiation, the photocleavable link to insulin is broken, and completely native and soluble insulin is released from the depot in a well behaved, first order fashion. These materials are 90% therapeutic, form completely soluble and injectable formulations in mildly acidic conditions, form insoluble depots at neutral pH, efficiently release soluble protein from these depots when irradiated, and leave behind only small easily absorbed molecules after irradiation. As such they approach ideality for photoactivated depot materials.

Dataset Information

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Publications

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Similar Datasets

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