1. RNDr. Jaroslav Turánek, CSc.
FT-IR spectroscopy for study of protein structure and protein temperature behaviour
RNDr. Jaroslav Turánek, CSc.
Výzkumný ústav veterinárního lékařství Brno
Farmakologický ústav LF MU

2. The amino acid sequence is called: Primary Structure
Proteins
Monomeric units of proteins: Amino acids
condensation reaction with amino acids (polymerization):
Product: POLYPEPTIDE CHAIN
The amino acid sequence is called: Primary Structure

3. Protein structure ß-sheet: ordered in layers random coil: chain
without periodic structure
-helix: screwed structure

4. FTIR for protein analysis
Secondary Structure Determination
random coil
Amid I :
C=O stretching vibration... υ δ
 - helix
 - sheet
...is sensitive to conformation

5. Secondary Structure Determination
Investigation of proteins by FT-IR
Secondary Structure Determination
Blue: Hemoglobine
74 % α-helix
0 % -sheet
Red: Concanavalin
46 % -sheet % α-helix
Just this single band (amide I) is used!!!

6. Investigation of proteins by FT-IR
Wasser
Water
Protein
Protein
20 mg/ml
Wasser
Protein
Proteinspektrum
Wasser
Protein
Proteinspektrum
Absorption of water and water vapor mask the protein signal
Problem:
requirements:
precise temperature (< 0,1K)
pathlength stability (< 0.1 %  7 nm)
sealed system

7. Investigation of proteins by FT-IR
transmission cell:
water soluble proteins
protein library
sensitive and sealed spectrometer
bio-ATR:
- membrane proteins
- biomolecular interactions
CONFOCHECK
dedicated software
temperature
control

8. Why FT-IR spectroscopy ?
Protein Analysis
Why FT-IR spectroscopy ?
Structure (conformation) is correlated to function
FT-IR spectroscopy is very sensitive to conformational changes !

9. CONFOCHECK: Applications
1) Fast determination of secondary structure
2) Detection of conformational changes
(pH, temperature)
3) Monitoring of biomolecular interactions
(protein-ligand binding) +

10. CONFOCHECK: Application
Determination of protein secondary structure
fast (within minutes!)
in native state (solution)
many buffer systems allowed
low amount of protein required

11. Protein Spectra Library
CONFOCHECK: Conformational analysis
Calibrated (PLS)
Protein Spectra Library
Secondary Structure Determination
Blue
74 % α-helix
0 % -sheet
Red
46 % -sheet % α-helix

12. CONFOCHECK: Conformational analysis
OPUS-Quant 2 for secondary structure determination
 - Helix content
Regression coefficient
R2 = 96,2
Error cross validation
RMSECV = 4,24
 - Sheet content
Regression coefficient
R2 = 95,1
Error cross validation
RMSECV = 3,19
Conformations from unknown protein structures are
predicted using PLS algorithm

13. CONFOCHECK: Protein library
Information block
Spectra search
30 proteins included

14. Biomolecular Interactions AquaSpecTM Transmission cell
FTIR for protein analysis
Protein Analysis
Biomolecular Interactions
Membrane/ Insoluble
Proteins
Soluble Proteins
Bio-ATR II
Bio-ATR I
AquaSpecTM Transmission cell
CONFOCHECK

15. CONFOCHECK: Water soluble proteins
AquaSpec-Transmission cell
Flow through cell with 6 µm path length
tubes of bio compatible high-grade steel
CaF2 windows
High pressure stability of path length (automated filling; HPLC!)
Bio compatible inline-filter included
Minimized volumes : 5 µl (including tubing and filters)

16. CONFOCHECK: Fast and easy analysis of proteins
Linear MCT detector
guaranteeing short
acquisition times
and excellent structure determination results
Beam path is shielded by special tubings (purgeable, easy exchangeable)
Extremely sealed
spectrometer
Accessories are recognized automatically (AARTM)

17. CONFOCHECK: Application
Detection of conformational changes
temperature pH
mutation
chemical influences

18. Temperature Induced Conformational Change
CONFOCHECK: Application
RNase
Detection of conformational changes
Temperature Induced Conformational Change
Temperature Induced Conformational Change
Red: 25°C
Light green: 35°C
Black: 45°C
Pink: 55°C
Red: 25°C
Light green: 35°C
Black: 45°C
Pink: 55°C
Dark green: 65°C
Blue: 75°C
Red: 25°C
Defolding of ß-sheet
FTIR is unique in detecting conformational
changes of complete proteins in aqueous solution

19. CONFOCHECK: Application
Temperature Induced Conformational Change
35°C
ß-Sheet Refolding
75°C
ß- Sheet Defolding
35°C
1500 cm-1
1700 cm-1
3d plot from differerence spectra