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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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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20 mg/ml

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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

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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

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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