Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.

TitleScreening of candidate substrates and coupling ions of transporters by thermostability shift assays.
Publication TypeJournal Article
Year of Publication2018
AuthorsMajd, H, King, MS, Palmer, SM, Smith, AC, Elbourne, LDh, Paulsen, IT, Sharples, D, Henderson, PJf, Kunji, ERS
JournalElife
Volume7
Date Published2018 10 15
ISSN2050-084X
KeywordsAdenosine Diphosphate, Adenosine Triphosphate, Animals, Biological Assay, Humans, Ions, Ligands, Membrane Transport Proteins, Mitochondria, Protein Stability, Reproducibility of Results, Substrate Specificity, Temperature, Tetrahymena
Abstract

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.

DOI10.7554/eLife.38821
Alternate JournalElife
Citation Key10.7554/eLife.38821
PubMed ID30320551
PubMed Central IDPMC6211832
Grant ListEM-2014 -045 / / Leverhulme Trust / International
JIF 062164/Z/00/Z / / Wellcome / International
MC_UU_00015/1 / / Medical Research Council / United Kingdom
MPSI BBS/B/14418 / / Biotechnology and Biological Sciences Research Council / United Kingdom
/ / Wellcome Trust / United Kingdom
JIF 062164/Z/00/Z / / Wellcome Trust / United Kingdom