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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
104
12-POS
Board 6
Structural Stability of Rhodopsin Analyzed by Single-Molecule Force Microscopy
Ya-Na Chen
1
, Maria Kamenetska
1
, Jacob Black
1
, Kelly J. Culhane
2
, Ziad Ganim
1
, Elsa C. Y.
Yan
1
.
1
Yale University, New Haven, CT, USA,
2
Yale University, New Haven, CT, USA.
Rhodopsin is a representative member of family A G-protein coupled receptors (GPCRs). Its
structural components include seven transmembrane helices and a chromophore molecule, 11-cis
retinal. As a biological dim-light detector, rhodopsin possesses exceptional photon-detecting
characteristics: high quantum yield and low dark noise. To elucidate how the structure of
rhodopsin give rise to its astounding biophysical properties, single-molecule force microscopy
(SMFM) experiments were carried out. Among different SMFM techniques, optical tweezers
(OT) is an ideal method due to its high force resolution and the versatility it offers in force
extension geometries. Being a membrane protein, to study rhodopsin
in vitro
, sample preparation
procedures such as protein engineering, expression and purification, have been carefully
optimized. A rhodopsin mutant has been designed to facilitate DNA linkage via cysteine-
maleimide covalent bonds at the N- and the C- termini. Rhodopsin-DNA handle complexes have
been successfully conjugated and characterized. Silica microspheres have been bio-
functionalized for the attachment of DNA handles through biotin / streptavidin or digoxigenin /
anti-digoxigenin interactions. Through optically trapping silica microspheres, force can thus be
applied to the entire assembled construct. Force-extension data presented display evidence for
reversible unfolding of rhodopsin. Analysis of more single-molecule force-extension traces will
provide a high molecular resolution model uncovering structure-function relations of rhodopsin.
Future studies involve altering protein environmental components to quantify effects on
rhodopsin structural stability. This study will not only impact and expand current understanding
of GPCRs but will also set the foundation for applying optical tweezers-based SMFM
experiments on a wide variety of transmembrane receptor, channel, and gate proteins.