Abstract
ABSTRACTImmunoglobulin (Ig) domains are the most prevalent protein domain structure and share a highly conserved folding pattern; however, this structural family of proteins is also the most diverse in terms of biological roles and tissue expression. Ig domains vary significantly in amino acid sequence but share a highly conserved tryptophan in the hydrophobic core of this beta-stranded protein. Palladin is an actin binding and bundling protein that has five Ig domains and plays an important role in normal cell adhesion and motility. Mutation of the core tryptophan in one Ig domain of palladin has been identified in a pancreatic cancer cell line, suggesting a crucial role for this sole tryptophan in palladin Ig domain structure, stability, and function. We found that actin binding and bundling was not completely abolished with removal of this tryptophan despite a partially unfolded structure and significantly reduced stability of the mutant Ig domain as shown by circular dichroism investigations. In addition, this mutant palladin domain displays a tryptophan-like fluorescence attributed to an anomalous tyrosine emission at 345 nm. Our results indicate that this emission originates from a tyrosinate that may be formed in the excited ground state by proton transfer to a nearby glutamyl residue. Furthermore, this study emphasizes the importance of tryptophan in protein structural stability and illustrates how tyrosinate emission contributions may be overlooked during the interpretation of the fluorescence properties of proteins.SHORT ABSTRACTThis study explores the functional and structural consequences of a point mutation in palladin, an Ig domain protein first identified in a pancreatic tumor cancer cell line. While exploring the consequences of mutating this conserved tryptophan in the hydrophobic core of the most prevalent domain structure found in proteins, an anomalous tyrosine fluorescence phenomenon was exposed.
Publisher
Cold Spring Harbor Laboratory