Conserved tryptophan mutation disrupts structure and function of immunoglobulin domain revealing unusual tyrosine fluorescence

Protein Sci. 2020 Oct;29(10):2062-2074. doi: 10.1002/pro.3929. Epub 2020 Sep 3.

Abstract

Immunoglobulin (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 341 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 aspartic acid 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.

Keywords: actin; immunoglobulin domains; protein folding; tryptophan; tyrosinate fluorescence.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Circular Dichroism
  • Immunoglobulin Domains
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / genetics*
  • Mice
  • Mutation, Missense*
  • Tryptophan / chemistry
  • Tryptophan / genetics

Substances

  • Immunoglobulin G
  • Tryptophan