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In Silico and In Vitro Considerations of Keratinocyte Nuclear Receptor Protein Structural Order for Improving Experimental Analysis

  • Rambon Shamilov
  • Matthew J. Staid
  • Brian J. AneskievichEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2109)

Abstract

Nuclear receptors (NR) regulate gene expression critical in keratinocyte replication and differentiation. In addition to a ligand-binding domain, NR like other transcription factor families have a DNA-binding domain that must attain a particular conformation for effective interaction with the three-dimensional structure in promoters of target genes for control of their expression. Such protein-DNA assemblies extend the classic “lock and key” idea typified by protein-protein interactions. However, it is becoming increasingly clear that multi-subdomain transcription factors like NR frequently range along the length of the protein from structured, ordered regions expected for interaction with a preset partner to more flexible, intrinsically disordered regions which are more available for diverse posttranslational modifications and/or interaction with differing partners. The extended amino terminus of NR (the A/B subdomain) is one such intrinsically disordered region. Here we provide a primer on in silico-based recognition of amino acid composition and order associated with such conformational flexibility along with adaptations of readily accessible laboratory techniques (e.g., considerations for recombinant expression, sensitivity to protease and proteasome digestion) to facilitate initial prediction and testing for intrinsic disorder in various proteins of interest to keratinocyte biologists, like NR and other transcription factors.

Keywords

Intrinsic disorder Protein conformation Transcription factor Nuclear receptor Keratinocyte 

Notes

Acknowledgments

Earlier work from our group referenced here was supported in part by a USPHS/NIH grant to BJA (AR048660) from NIAMS. RS was supported by an assistantship from the Department of Pharmaceutical Sciences.

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Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • Rambon Shamilov
    • 1
  • Matthew J. Staid
    • 2
  • Brian J. Aneskievich
    • 3
    Email author
  1. 1.School of PharmacyUniversity of ConnecticutStorrsUSA
  2. 2.School of PharmacyUniversity of ConnecticutStorrsUSA
  3. 3.Department of Pharmaceutical Sciences, School of PharmacyUniversity of ConnecticutStorrsUSA

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