1. a useful tool to probe protein structural changes
Raman spectroscopy:
a useful tool to probe protein structural changes
Michele Di Foggia, Paola Taddei,
Sergio Bonora, Anna Tinti
Dip. Scienze Biomediche e Neuromotorie - Università di Bologna
Armida Torreggiani
ISOF - CNR, Bologna
Now, we want to insist on a special point:…. 1

2. Raman Spectroscopy
IT’S A LASER VIBRATIONAL SPECTROSCOPY (I.E. FT-IR) CHARACTERISED BY:
- no sample preparation, non destructive technique;
- unaffected by water and glass;
- coupled to a microscope, very high spatial resolution;
- can give quantitative results;
- provides a fingerprint (molecular identity) by which the molecule can be identified;
- extremely sensitive to stuctural changes. =

3. Raman spectroscopy : Applications
Raman spectroscopy has been successfully applied to the analysis of a wide-ranging number of systems.
Materials
Cultural Heritage
Pharmaceutical Science
Biomedical Science
Protein science

4. Raman spectroscopy can be useful: for monitoring processes
Callendar et al., Annu. Rev. Biophys. Biomol. Struct. 1994
for giving
a picture of
the binding
mode between
ligand and protein

5. -GLY-ALA-SER-ASP-PHE-VAL-TYR-CYS
for identifing radical-induced modifications on protein structure
Primary
structure X• X •
-helix
-sheet
Secondary
structure
Tertiary
structure
-GLY-ALA-SER-ASP-PHE-VAL-TYR-CYS
Multitude of
possible sites
of attack
(i.e. on AA side chains)
A wide range of different radicals can be formed 5

6. Secondary structure Amide I Amide III
-helix
-sheet
Secondary structure
The frequency of the vibrational modes of peptide bonds (Amide I and III) reflects the structure of the polypeptide chain
Amide I
Amide III
-helix
-sheet
Random Coil
Wavenumber / cm-1
1680
1660
1640
1300
1260
1220
Vibrational modes of
amide I
and
amide III
Amide I and III
arise from the vibration of the peptide carbonyl group 6

7. Local environment of side chains
affects the position and the intensity of Raman marker bands
Tyr residue
Trp residue
Aromatic residues
Marker bands
sensitive
to the environment
Phe residue
His residue
Tuma, J. Raman Spectrosc. 36 (2005) 307
Tuma and Thomas, Handbook of Vibrational Spectroscopy, 23 (2002) 215.

8. Secondary structure Amide I Amide III Trp Phe Tyr S-S Met C-S Trp Phe
(Peptide bond vibrations)
Amide I
Amide III
1600
1400
1200
1000
800
600
400
Raman spectrum of Lysozyme
Trp
Phe
Tyr
S-S
Met
C-S
Trp
Phe
Tyr
S-S
Met

9. Thermal aggregation of bovine serum albumin (BSA)
Heat
BSA
Tyr residues play an important role in the formation of the inter-molecular interactions
buried
exposed
Conversion of
-helices into -sheets
Navarra et al, J. Inorg. Biochem (2009) 1729
Jurasekova, Tinti, Torreggiani, Anal. Bioanal. Chem (2011) 2921

10. Metal coordination Cys His Zn ggg 500-515 cm-1 ggt 520-525 cm-1 tgt
Takeuchi, Biopolymers 72 (2003) 305
Torreggiani et al., J. Raman Spectrosc. 57 (2000) 149

11. BLG Cu-BLG Zn-BLG Cu-BLG Zn-BLG BLG BLG
Influence of metal ions on BLG thermal aggregation
Metal coordination
BLG
Cu-BLG
Cu-BLG
Zn-BLG
Zn-BLG
BLG
Cu2+ and Zn2+ promote a faster fibril formation,
but Zn2+ ions induce bigger aggregates.
Navarra, Tinti, Di Foggia, Leone, Militello, Torreggiani, J. Inorg. Biochem (2014) 64 11

12. ? Proteins Where is the main “target” -GLY-ALA-SER-ASP-PHE-VAL-TYR-CYS
Primary
structure X• X • •
Secondary
structure
-helix
-sheet
-GLY-ALA-SER-ASP-PHE-VAL-TYR-CYS
Where is the main “target” ?
i.e.
Multitude of possible sites of attack 12

13. Metallothioneins
MTs undergone to .H and eaq- attack
Zn-MT
Cd-MT
metal-Cys
metal-Cys
Met
S-S
Met
0 Gy
100 Gy
50 Gy
Radical attack towards metal-Cys clusters and Met: induces structural rearrangements (especially in Zn-MT).
Torreggiani, et al., Chem. Eur. J. 15 (2009) 6015 13

14. - thermal aggregation; - presence of metal ions;
Conclusions
Raman spectroscopy has been proved to be extremely useful to obtain structural information changes induced by different stimuli:
- thermal aggregation;
- presence of metal ions;
- damages induced by free radical stress;
- thermal or chemical denaturation;
- adsorption on biomedical devices/biomimetic surfaces. 14

15. Looking forward your questions and collaboration proposals15