Format

Send to

Choose Destination
J Mol Biol. 2014 Oct 23;426(21):3670-80. doi: 10.1016/j.jmb.2014.08.020. Epub 2014 Aug 29.

Characterization of membrane protein interactions by isothermal titration calorimetry.

Author information

1
Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA 90033, USA.
2
Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA 90033, USA. Electronic address: tulmer@usc.edu.

Abstract

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of -4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500±100s(-1) and 2.1±0.1s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.

KEYWORDS:

NMR spectroscopy; isothermal titration calorimetry; membrane proteins; protein–protein interaction; transmembrane association

PMID:
25178257
DOI:
10.1016/j.jmb.2014.08.020
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center