Use of multidimensional fluorescence resonance energy transfer to establish the orientation of cholecystokinin docked at the type A cholecystokinin receptor

Kaleeckal G. Harikumar, Fan Gao, Delia I. Pinon, Laurence J. Miller

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Fluorescence resonance energy transfer (FRET) represents a powerful tool to establish relative distances between donor and acceptor fluorophores. By utilizing several donors situated in distinct positions within a docked full agonist ligand and several acceptors distributed at distinct sites within its receptor, multiple interdependent dimensions can be determined. These can provide a unique method to establish or confirm three-dimensional structure of the molecular complex. In this work, we have utilized full agonist analogues of cholecystokinin (CCK) with Aladan distributed throughout the pharmacophore in positions 24, 29, and 33, along with receptor constructs derivatized with Alexa546 at positions 94, 102, 204, and 341 in the helical bundle and first, second, and third extracellular loops, respectively. These provided 12 FRET distances to overlay on working models of the CCK-occupied receptor. These established that the carboxyl terminus of CCK resides at the external surface of the lipid bilayer, adjacent to the receptor amino-terminal tail, rather than being inserted into the helical bundle. They also provide important experimentally derived constraints for understanding spatial relationships between the docked ligand and the flexible extracellular loop regions. Multidimensional FRET provides a new independent method to establish and refine structural insights into ligand-receptor complexes.

Original languageEnglish (US)
Pages (from-to)9574-9581
Number of pages8
JournalBiochemistry
Volume47
Issue number36
DOIs
StatePublished - Sep 9 2008

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Use of multidimensional fluorescence resonance energy transfer to establish the orientation of cholecystokinin docked at the type A cholecystokinin receptor'. Together they form a unique fingerprint.

Cite this