Engineering High-Refractive-Index Proteins Inspired by Lens Crystallins
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vanderHilst-jelle-phd-be-2025-thesis.pdf
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Author(s)
van der Hilst, Jelle Dirk
Advisor(s)
Voigt, Christopher A.
Belcher, Angela M.
Date Issued
May 2025
Publisher
Massachusetts Institute of Technology
Abstract
Lens proteins called crystallins form transparent and volume-spanning hydrogels which are able to refract incident light. The unique properties of these proteins include expression at concentrations up to 1000 mg/mL, dense packing into lenses with high refractive indices, and a lifelong stability without the option of renewal and turnover. Crystallins even form GRadient INdex (GRIN) lenses, sophisticated optical materials which exhibit significant benefits over classical lenses but are challenging to produce with abiotic techniques.
In this dissertation, we detail efforts to mine, express, and characterize S-crystallins from powerful cephalopod GRIN lenses. We observe a small S-crystallin, Lops4, to self-assemble into 50-70 nanometer anisotropic particles, though it—and 12 other S-crystallins tested—is prone to precipitation and poorly soluble in lab conditions, frustrating attempts to form protein-polymer hydrogel lens materials. We demonstrate the formation of synthetic GRIN materials with another soluble protein, bovine serum albumin (BSA), via iterative casting of poly(acrylamide) layers. We then turned to the large protein sequence database UniProt to computationally screen for other natural sequences with high refractive increments, theorizing that there must be natural sequences with higher refractive increments and more forgiving biophysical properties than the S-crystallins. Indeed, 44 proteins were identified with refractive increments higher than any previously published. The top hits were synthesized and expressed, with several displaying meaningful expression levels and high measured refractive increments. Finally, we set out to demonstrate full control over the refractive increment of the highly soluble protein SUMO at the sequence level. Mutations were computationally predicted using PyRosetta to raise or lower refractive index without affecting stability or solubility. 36 of these proteins were designed with a range of refractive increments spanning nine standard deviations from the mean protein refractive increment. While expression of these proteins is decreased proportionally with the number of mutations, several variants demonstrate tailorable refractive index versus wild type.
This work compares alternative approaches to making synthetic crystallins, and highlights the challenges of working with proteins whose function requires both a high refractive increment and solubility at extremely high concentrations. The benefits of protein components – a high achievable range of refractive index unmatched by most abiotic materials, cheap and sustainable scaling, and the ability to easily and non-monotonically pattern proteins and thus refractive index within a material – will contribute to the construction of the optical materials of the future.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
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