Format

Send to

Choose Destination
See comment in PubMed Commons below
Biochemistry. 2016 Feb 16;55(6):850-9. doi: 10.1021/acs.biochem.5b01268. Epub 2016 Feb 5.

Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles.

Author information

  • 1Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University (WCMC) , New York, New York 10065, United States.
  • 2Computational Chemistry and Molecular Biophysics Unit, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health , Baltimore, Maryland 21224, United States.
  • 3Division of Molecular Therapeutics, New York State Psychiatric Institute , New York, New York 10032, United States.
  • 4HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University , New York, New York 10065, United States.

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.

PMID:
26811944
PMCID:
PMC4757857
DOI:
10.1021/acs.biochem.5b01268
[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society Icon for PubMed Central
    Loading ...
    Support Center