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

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. =

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

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

-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

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

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.

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

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. 103 (2009) 1729 Jurasekova, Tinti, Torreggiani, Anal. Bioanal. Chem. 400 (2011) 2921

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

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. 137 (2014) 64 11

? 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

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

- 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

Looking forward your questions and collaboration proposals 15