Secondary structure of single protein using SERS

Protein conformations are usually examined on a set of molecules
by circular dichroism or by infrared or Raman spectroscopy. Spectrum analysis
then requires a deconvolution of the bands to determine the secondary structures of the
proteins. Here, we propose to use Raman Spectroscopy enhanced by
gold nanoparticles (SERS) giving access to the single molecule in order to determine the
structures of individual proteins interacting with gold.
We studied bovine serum albumin (BSA) protein in the vicinity or in contact with
gold nanoparticles 80 nm in diameter. Plasmonic exaltation allowed us to
determine conformations at the single protein scale. 500,000 SERS spectra of
individual proteins were then statistically analyzed for different
conditions of pH (between pH=2 and pH=7) and temperature (between 22° and 55°C).
The results show that the structure of each individual protein can be identified
optically by its amide bands I, II and III providing information on the α helical forms or the
β1, β2 and βT sheets. The diffusion of the bands observed at the microscopic scale on the
chromatograms explains the broadening of classical Raman bands. BSA at pH
physiological and room temperature (22°C) having a globular shape has less tendency
to adsorb on gold. On the other hand, as the temperature increases or the pH decreases,
BSA performs a conformational transition to a cigar shape .
SERS is therefore a new technique that allows the determination of both the kinetics
adsorption but also structural variations of individual proteins. The SERS allows
also to avoid the complex fitting of often inextricable spectra due to the
diversity of secondary structures

This work has been supported by the European Regional Development Fund (FEDER) and the Region Bourgogne Franche Comté under the project "NanoNeuromed".

Outline :
00:00 1. Introduction
01:17 2. Methods to determine the protein structure(MS, SPM, NMR, RX, FTIR, Raman)
06:08 3. Experimental set-up
07:29 4. Fluctuation of Amides at Ph 7 and ambiant temperature
11:18 5. Effect of the pH
16:13 6. Effect of the Temperature
18:47 7. Conclusion