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Conformational Changes in Palladin Actin‐Binding Domains Measured by Fluorescent Resonance Energy Transfer
Author(s) -
Womack Samuel,
Vattepu Ravi,
Beck Moriah Rene
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.762.10
Subject(s) - förster resonance energy transfer , actin remodeling , actin binding protein , mdia1 , actin , microbiology and biotechnology , biophysics , chemistry , fluorescence recovery after photobleaching , actin remodeling of neurons , microfilament , cytoskeleton , context (archaeology) , actin cytoskeleton , fluorescence microscope , biology , biochemistry , fluorescence , cell , physics , quantum mechanics , paleontology , membrane
The fundamental goal of our research is to understand how cell motility in both normal and metastatic cells is regulated by palladin, an actin binding protein that plays a key role in remodeling the actin cytoskeleton. Our recent work has established that palladin regulates actin dynamics and organization by enhancing the polymerization rate and stabilizing filaments in an orthogonal meshwork. Palladin family members all utilize immunoglobulin‐like domains to bind and crosslink actin filaments. Various isoforms of palladin contain from three to five immunoglobulin‐like domains that have been implicated in a variety of protein‐protein interactions. Our previous results indicate that the Ig3 domain of palladin is directly involved in binding and crosslinking of actin and also promotes actin polymerization, whereas the Ig4 domain of palladin does not interact with actin directly. Nevertheless a tandem Ig3–4 domain protein exhibits dramatically more efficient actin binding, crosslinking, and polymerization than the isolated Ig3 domain. Recently we established that the Ig3–4 domains of palladin undergo actin‐induced dimerization, which likely results in a conformational change. NMR chemical shift measurements indicate that interactions between the tethered Ig3 and Ig4 domains are limited to the interface and lead to no more than marginal structural changes. To further narrow the gap in our understanding of palladin's role in actin assembly, we need to examine these interactions in the cellular context and demonstrate that palladin can bind directly to monomeric actin. Our current efforts are aimed at combining fluorescence resonance energy transfer (FRET) and confocal microscopy to analyze the interactions of palladin and G‐actin within cells and with isolated proteins. These experiments will also enable us to investigate the dynamic intra‐ and intermolecular interactions through the use of sophisticated fluorescent probes. Our first goal was to use site‐directed mutagenesis to replace six cysteines with alanine in two tandem immunoglobulin‐like domains of palladin before confirming that these mutations do not interfere with actin binding and bundling functions of palladin. We will share our results that make use of a newly synthesized Pacific Blue fluorescent trivalent nickel complex that we attach to the C‐terminal His 6 ‐purification tag on our palladin constructs. We have also introduced a unique cysteine residue at the N‐terminus of palladin to allow conjugation to fluorescein maleimide so that we can track conformational changes that occur in the tandem actin‐crosslinking domains of palladin using FRET. Support or Funding Information This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418 and P20 GM103638 and an undergraduate research grant from Wichita State University.