RNDr. Jaroslav Turánek, CSc.

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Presentation transcript:

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

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

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

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

Secondary Structure Determination 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!!!

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

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

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 !

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

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

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

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

CONFOCHECK: Protein library Information block Spectra search 30 proteins included

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

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)

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)

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

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

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