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What's linker got to do with it? Examining the mechanical stability of palladin's Ig3-4 linker region
Hughes, Lauren Sargent, Rachel Bradford, Colby Ta, Nathan H.
Hughes, Lauren
Sargent, Rachel
Bradford, Colby
Ta, Nathan H.
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2026-04-24
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Hughes, Lauren M.; Sargent, Rachel A.; Bradford, Colby W.; Ta, Nathan H.; Beck, Moriah R. 2026. What's linker got to do with it? Examining the mechanical stability of palladin's Ig3-4 linker region. -- In Proceedings: 25th Annual Undergraduate Research and Creative Activity Forum. Wichita, KS: Wichita State University.
Abstract
Actin is the most abundant protein within all eukaryotic cells and is essential for motility, structure, and cellular division. Among actin’s many binding partners is palladin, a lesser-known protein that is required for embryonic development and is highly expressed in metastatic cancer cells. Understanding the function of paladin and its role in cancer metastasis requires structural analysis. Palladin is comprised of five immunoglobulin-like domains (Ig), each connected via a flexible linker region. Previous research has shown that Ig3 is the minimal actin-binding domain; however, the binding affinity and bundling are significantly increased when the Ig3-4 linker domain is present. The Beck Lab introduced several mutations to the linker to explore the effects of linker length, charge, and residue patterning. The most prominent mutation is RLinkerA, a conversion of the domain’s ten arginines into alanines, which completely disrupted the actin-binding ability of palladin. All mutated and wild-type linker regions underwent circular dichroism spectroscopy to determine secondary structure, as well as chemical and thermal denaturation tests to determine stability. These tests indicate that the secondary structure is not altered among the mutations, proving that the lack of binding affinity in RLinkerA is not due to a catastrophic unfolding event. Additionally, the denaturation data indicated that the RLinkerA mutation has a similar stability to wild-type. Together, these results show that both the net charge and sequence order of the linker are critical for modulating palladin’s actin-binding and bundling activity. Our future studies seek to understand the stability of palladin in a dynamic, mechanical environment. To achieve this, we will use optical tweezers to stimulate muscle pulls on wild-type and mutant palladin. Determining the mechanical stability of the Ig3-4 linker region is essential for determining how palladin participates in binding and bundling interactions with actin, and ultimately, how palladin contributes to cancer cell metastasis.
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Presented to the 25th Undergraduate Research and Creative Activity Forum (URCAF) held in Woolsey Hall, Wichita State University, April 24, 2026.
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Wichita State University
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URCAF;v.25