Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session II
48 – POS
Board 20
The Knott and Chromophore of Near-infrared Fluorescent Probe iRFP: Impact on Protein
Folding
Olesya V. Stepanenko
1
, Gregory S. Bublikov
1
, Olga V. Sepanenko
1
, Vladislav V.
Verkhusha
2
,
Konstantin K. Turoverov
1,3
, Irina M. Kusnetsova
1,3
.
1
Institute of Cytology RAS, St.Petersburg, Russian Federation,
2
Albert Einstein College of
Medicine, USA, New York, NY, USA,
3
St Petersburg State Polytechnical University,
St.Petersburg, Russian Federation.
The near-infrared fluorescent probes engineered from bacterial phytochrome photoreceptors
(BphPs) make an advance in non-invasive imaging of biological processes in vivo. These
proteins are able to develop the near-infrared fluorescence binding and utilizing as a
chromophore the biliverdin IXa (BV) available in the mammalian cells. BphPs and their
derivative probes contain a unique figure-of-eight knot between domains involved in
chromophore binding. The BphPs knot is supposed to be important for chromophore binding and
for transduction of conformational changes induced by light absorption by the chromophore to
the effector domain of photoreceptors. Study of knotted proteins is of high importance for
solving the fundamental problem of protein folding.
We investigated the influence of chromophore and knot on the folding of the near-infrared iRFP
developed from BphPs, RpBphP2. To this aim we obtained and characterized iRFP in its
holoform (in complex with the chromophore) and its apoform (chromophore-free). The spectral
features of iRFP in the apoform suggest that it retains the secondary and tertiary structures
inherent to the holoform of protein. Apoprotein incorporates BV at a molar ratio of 1:1 as
indicated by equilibrium microdialysis. The conformation and microenvironment of BV in the
apo-iRFP/BV complex was similar to that for native iRFP. Denaturation – renaturation
experiments on iRFP in holoform and apoform revealed that the knot does not prevent efficient
protein folding while the chromophore stimulates protein aggregation and thus inhibits protein
refolding.
This work was supported by Program MCB RAS, grant RFBP (13-04-01842) and scholarships of
the President of RF (SP-65.2012.4 and SP-563.2012.4).
- 93 -
