1 Toxic Effects of Pristine and Carboxyl Functionalized Graphene on p53 Protein CASE STUDY

Size of Different Objects Bhasma, Metal ion (Cd, Pb etc.) In Ayurvedic Medicine

DNA Damage Cell Cycle Arrest Hypoxia Cell Cycle Arrest DNA Repair Cell Cycle Restart Apoptosis and Senescence Death & elimination of damaged cells Guardian of Genome P53MDM2 Active form P53 Angiogenesis Cellular and Genetic Stability

4 How graphene and its functionalized form (COOH) interact with p53 tumor suppressor protein? A Computational Nanotoxicology study.

Structure of Human p53 protein in complex with 1 nm Graphene p53 _grap1nm  -12 Kcal/mol Crystal structure of Human p53 protein BINDING STUDIES OF GRAPHENE OF SIZE 1 nm, 6 nm and 10 nm WITH HUMAN p53 TETRAMER How graphene of different sizes effect the normal functioning of the p53 protein?

BE  kcal/mol General Mode of binding of human p53 tetramer and DNA Mode of binding of human p53 tetramer with Graphene of 1 nm size and DNA Mode of Binding of Graphene of 1 nm size do not have any influence on p53- DNA complexes. Interestingly this preliminary result suggest that Graphene of 1 nm size can be used as a Drug delivery agent.

p53_grap6nm  Kcal/mol p53_grap6nm_DNA  Kcal/mol A-chain π-π perpendicular stacking (2.5 Å) interaction of Graphene with Phe212 residue of p53 Mechanism of Binding of p53 and Graphene of size 6 nm (a) and p53-graphene (6 nm)- DNA complexes (b) (a) (b)

TargetLigand Binding energy (Kcal/mol) 5 Å ligand interacting residues p53 (2OCJ- human p53 crystal structure ) Graphene 6 nm A Chain: SER 99, LYS 101, ARG 158, LEU 206, ASP 208, SER 261, ASN 263, LEU 264, ARG 267 C Chain: GLN 167, HIS 168, THR 170, GLU 171, PRO 177, ARG 181, PHE 212, MET 243, ASN 247, ARG 249 Human p53 tetramer- Graphene 6 nm DNA B Chain: TYR 107, SER 149, THR 150, PRO 151, PRO 152, ARG 202, GLU 221, GLU 224, VAL 225 C Chain: PRO 177, GLU 180, ARG 181, SER 185, ASP 186, LEU 188, PRO 199, GLN 192, LEU 201, ARG 202, VAL 203, PHE 212 Binding energy and interacting amino acids of p53-graphene 6 nm complexes and also its interaction with DNA The interaction (binding) energy, the binding pattern and the dynamic conformational changes of the p53-graphene (6 nm) complexes revealed potential interference of graphene with the functional aspects of p53.

A-chain C-chain B-chain D-chain Human p53 tetramer interaction with 5% COOH group functionalized graphene of 6 nm size BE  kcal/mol

CONCLUSIONS Normal DNA repair mechanism -- DNA makes contact with four domains (A, B, C and D) of p53 protein. Possible effects of graphene of different sizes on the molecular structure and function of p53 revealed. 1 nm pristine and COOH functionalized graphene sheets did not interfere with the normal p53 functioning. Pristine graphene of size 6 nm (and its COOH functionalized sheet) and 10 nm blocks the binding of DNA to two domains (A and D) of p53. This indicates the graphene intervention with the DNA binding mechanism and thereby halting the DNA repair mechanism  Tumorigenesis (graphene toxicity). This may be due to the strong adsorbing property of graphene sheet that pulls the DNA towards itself restricting DNA’s interaction towards p53’s A and D-chains.

Acknowledgements Director, Amrita Centre for Nanosciences and Molecular Medicine, Kochi, Kerala State Dr. Shikhar Gupta, NIPER M.Tech. Students: Ph.D. Students: Faiza B, Sneha P, Shruthy K, Ms. Anju CP, ACNSMM Shraddha P, Aathira B, Geetha P Ms. Jane Jose, ACNSMM Ms. Anu R. Melge, JRF, ACNSMM Indo-Finland Collaborators: Dr. Adyary Fallareo and Prof. Pia Vuorela University of Helsinki, Finland 11 Department of Biotechnology (DBT) - India under Nanotoxicology and Nanomedicine-Phase-II Program to ACNSMM

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