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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. - PowerPoint PPT Presentation
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RNDr. Jaroslav Turánek, CSc.Výzkumný ústav veterinárního lékařství Brno
Farmakologický ústav LF MU
FT-IR spectroscopy for study of protein structure and protein
temperature behaviour
Monomeric units of proteins: Amino acids
Proteins
The amino acid sequence is called: Primary Structure
condensation reaction with amino acids (polymerization):
Product: POLYPEPTIDE CHAIN
ß-sheet: ordered in layers
-helix: screwed structure
Protein structure
random coil: chain without periodic structure
Amid I :
C=O stretching vibration...
N
O
H
υ
δ
FTIR for protein analysis
...is sensitive to conformation
random coil
- helix
- sheet
Secondary Structure Determination
Sample Name 20mg/ml Concanavalin A in 20 mmol KH2PO4 pH7
Sample Name 20mg/ml Myoglobin in 20 mmol KH2PO4 pH7
120013001400150016001700
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Abs
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Uni
tsInvestigation of proteins by FT-IR
Secondary Structure DeterminationBlue: Hemoglobine
74 % α-helix
0 % -sheet
Red: Concanavalin46 % -sheet 0 % α-helix
Just this single band (amide I) is used!!!
1200130014001500160017001800
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Protein
20 mg/ml
12001400160018002000
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Proteinspektrum
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Proteinspektrum
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Water
Protein
Investigation of proteins by FT-IR
Absorption of water and water vapor mask the protein signal
Problem:
requirements: precise temperature (< 0,1K)
sealed system
pathlength stability (< 0.1 % 7 nm)
sensitive and sealed spectrometer
Investigation of proteins by FT-IR
dedicated softwaretemperaturecontrol
transmission cell:
water soluble proteins
bio-ATR:- membrane proteins
- biomolecular interactions
protein library
Protein Analysis
Why FT-IR spectroscopy ?
Structure (conformation) is correlated to function
FT-IR spectroscopy is very sensitive to
conformational changes !
2) Detection of conformational changes
(pH, temperature)
1) Fast determination of secondary structure
CONFOCHECK: Applications
3) Monitoring of biomolecular interactions
(protein-ligand binding)
+
Determination of protein secondary structure
• fast (within minutes!)
• in native state (solution)
• many buffer systems allowed
• low amount of protein required
CONFOCHECK: Application
Sample Name 20mg/ml Concanavalin A in 20 mmol KH2PO4 pH7
Sample Name 20mg/ml Myoglobin in 20 mmol KH2PO4 pH7
120013001400150016001700
Wavenumber cm-1
0.00
00.
005
0.01
00.
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0.02
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Abs
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Secondary Structure DeterminationBlue
74 % α-helix
0 % -sheet
Red 46 % -sheet 0 % α-helix
Calibrated (PLS)
Protein Spectra Library
CONFOCHECK: Conformational analysis
OPUS-Quant 2 for secondary structure determination
CONFOCHECK: Conformational analysis
- Helix content
Regression coefficientR2 = 96,2
Error cross validationRMSECV = 4,24
- Sheet content
Regression coefficientR2 = 95,1
Error cross validationRMSECV = 3,19
Conformations from unknown protein structures are predicted using PLS algorithm
CONFOCHECK: Protein library
R:\Software\Bruker Protein Library\Bib_spec\a-Chymotrypsin_bib.1 a-Chymotrypsin | Av. of 3 6 µm transmission cell, 25°C, 18 mg/ml a-Chymotrypsin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Albumin_bib.1 Albumin | Av. of 3 6 µm transmission cell, 25°C, 15.0 mg/ml bovine serum Albumin in 40 mmol phosphate buffer pH 7 R:\Software\Bruker Protein Library\Bib_spec\Aldolase_bib.1 Aldolase | Av. of 3 6 µm transmission cell, 25°C, 20,8 mg/ml Aldolase from rabbit muscle in 40 mmol KH2PO4, pH 7 R:\Software\Bruker Protein Library\Bib_spec\Carbonic_Anhydrase_bib.1 Carbonic Anhydrase | Av. of 3 6 µm transmission cell, 25°C, 19.33 mg/ml Carbonic Anhydrase in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Catalase_bib.1 Catalase | Av. of 3 6 µm transmission cell, 25°C, 20 mg/ml Catalase in aqueous solution pH7 R:\Software\Bruker Protein Library\Bib_spec\ConcanavalinA_bib.1 Concanavalin A | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Concanavalin A in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Cutinase_bib.1 Cutinase_25-45-25 20 mmol KH2PO4, pH 6 R:\Software\Bruker Protein Library\Bib_spec\Glucose_oxidase_bib.1 Glucose Oxidase | Av. of 3 6 µm transmission cell, 25°C, 18.06 mg/ml Glucose Oxidase in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Glutamic-Oxalacetic Transaminase_bib.1 Glutamic-Oxalacetic Transaminase | Av. of 3 6 µm transmission cell, 25°C, 20 mg/ml Glutamic-Oxalacetic Transaminase from porcine heart in 40 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Hemoglobin_bib.1 Hemoglobin | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Hemoglobin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Human_IgG_bib.1 Human Immunoglobulin | Av. of 3 6 µm transmission cell, 25°C, 19 mg/ml Human Immunoglobulin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Lectin_bib.1 Lectin | Av. of 3 6 µm transmission cell, 25°C, 21 mg/ml Lectin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Lysozym_bib.1 Lysozym | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Lysozym in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Myoglobin_bib.1 Myoglobin | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Myoglobin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Papain_bib.1 Papain | Av. of 3 6 µm transmission cell, 25°C, 15mg/ml Papain in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Pepsinogen_bib.1 Pepsinogen | Av. of 3 6 µm transmission cell, 25°C, 19,4 mg/ml Pepsinogen in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Pepsin_bib.1 Pepsin | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Pepsin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Protease_bib.1 Protease | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Protease in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Ribonuclease_A_bib.1 Ribonuclease A | Av. of 3 6 µm transmission cell, 25°C, 24 mg/ml Ribonuclease A in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Ribonuclease_S_bib.1 Ribonuclease S | Av. of 3 6 µm transmission cell, 25°C, 18 mg/ml Ribonuclease S in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Superoxid Dismutase_bib.1 Superoxid Dismutase | Av. of 3 6 µm transmission cell, 25°C, 22 mg/ml Superoxid Dismutase in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Transaldolase_bib.1 Transalolase | Av. of 3 6 µm transmission cell, 25°C, 20 mg/ml Transalolase from bakers yeast 40 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Trypsinogen_bib.1 Trypsinogen | Av. of 3 6 µm transmission cell, 25°C, 20mg/ml Trypsinogen in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Trypsin_bib.1 Trypsin | Av. of 3 6 µm transmission cell, 25°C, 20 mg/ml Trypsin in 20 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\Ubiquitin_bib.1 Ubiquitin | Av. of 3 6 µm transmission cell, 25°C, 20 mg/ml Ubiquitin in 40 mmol phosphate buffer pH7 R:\Software\Bruker Protein Library\Bib_spec\ß-Lactoglobulin_bib.1 ß-Lactoglobulin | Av. of 3 6 µm transmission cell, 25°C, 15.8 mg/ml ß-lactoglobulin in 40 mmol phosphate buffer pH 7
28/09/200023/10/200108/01/200127/10/200028/09/200025/09/200027/09/200109/01/200125/09/200028/09/200028/09/200025/09/200025/09/200025/09/200028/09/200025/09/200025/09/200028/09/200028/09/200028/09/200009/01/200125/09/200025/09/200008/01/200123/10/2001
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Information block
Spectra search
30 proteins included
FTIR for protein analysis
Soluble Proteins
AquaSpecTM Transmission cell
Bio-ATR IBio-ATR II
Biomolecular Interactions
Membrane/ InsolubleProteins
AquaSpec-Transmission cell
Bio compatible inline-filter included
Flow through cell with 6 µm path length
CaF2 windows
tubes of bio compatible high-grade steel
Minimized volumes : 5 µl (including tubing and filters)
High pressure stability of path length (automated filling; HPLC!)
CONFOCHECK: Water soluble proteins
CONFOCHECK: Fast and easy analysis of proteins
Extremely sealed
spectrometer
Accessories are recognized
automatically (AARTM)
Beam path is shielded by special
tubings (purgeable, easy
exchangeable)
Linear MCT detector
guaranteeing short
acquisition times
and excellent structure
determination results
Detection of conformational changes
CONFOCHECK: Application
pH
chemical
influences
temperature
mutation
1400145015001550160016501700
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Red: 25°C
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Red: 25°CLight green: 35°CBlack: 45°CPink: 55°C
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Red: 25°CLight green: 35°CBlack: 45°CPink: 55°CDark green: 65°CBlue: 75°C
Temperature Induced Conformational Change
Detection of conformational changes
CONFOCHECK: Application
RNaseTemperature Induced Conformational Change
Defolding of ß-sheet
FTIR is unique in detecting conformational changes of complete proteins in aqueous solution
3d plot from differerence spectra
1500 cm-1 1700 cm-1
35°C
75°C
35°C
ß-Sheet Refolding
ß- Sheet Defolding
Temperature Induced Conformational Change
CONFOCHECK: Application