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RNDr. Jaroslav Turánek, CSc. Výzkumný ústav veterinárního lékařství Brno

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. condensation reaction with amino acids (polymerization):. Product: POLYPEPTIDE CHAIN. Proteins.

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RNDr. Jaroslav Turánek, CSc. Výzkumný ústav veterinárního lékařství Brno

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

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

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

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

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

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

  8. Why FT-IR spectroscopy ? Protein Analysis 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 systemsallowed • low amount of protein required

  11. CONFOCHECK: Conformational analysis Calibrated (PLS) Protein Spectra Library Secondary Structure Determination Blue 74 % α-helix 0 % -sheet Red 46 % -sheet 0 % 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. 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. Beam path is shielded by special tubings (purgeable, easy exchangeable) CONFOCHECK: Fast and easy analysis of proteins Linear MCT detector guaranteeing short acquisition times and excellent structure determination results Extremely sealed spectrometer Accessories are recognized automatically (AARTM)

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

  18. Red: 25°C Temperature Induced Conformational Change Defolding of ß-sheet 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 RNase CONFOCHECK: Application Temperature Induced Conformational Change Detection of conformational changes 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

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