Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles. Academic Article uri icon

Overview

abstract

  • Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins.

publication date

  • February 5, 2016

Research

keywords

  • Amino Acid Transport Systems
  • Detergents
  • Leucine
  • Lipids
  • Micelles

Identity

PubMed Central ID

  • PMC4757857

Scopus Document Identifier

  • 84958824982

Digital Object Identifier (DOI)

  • 10.1038/nature07063

PubMed ID

  • 26811944

Additional Document Info

volume

  • 55

issue

  • 6