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

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.

SUBMITTER: Vattepu R

PROVIDER: S-EPMC7513720 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.

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

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