Bioinformatics: Practical Application of Simulation and Data Mining Protein Aggregation I Prof. Corey O’Hern Department of Mechanical Engineering & Materials Science Department of Physics Yale University 1

Langevin Dynamics 2

3 J. D. Honeycutt and D. Thirumalai, “The nature of folded states of globular proteins,” Biopolymers 32 (1992) 695. “Algorithms for brownian dynamics,” Molecular Physics 45 (1982) 637

Protein Folding Problem  Protein Aggregation Problem Which amino acid sequences fold to unique 3D structures and are not prone to aggregation? 4 Scenarios for protein lifespan

Role of Folded Intermediates 5

Protein Aggregation Self-assembled structures that grow into large, insoluble aggregates Aggregation more likely for unfolded/partially folded protein; exposed hydrophobic regions on separate monomers bind Aggregation caused by protein overproduction, stress, mutation Variables that affect aggregation: amino acid sequence, environmental factors such as pH, temperature, protein concentration, chaperones Types of aggregates: amyloid fibrils (in vivo, in vitro, rich in beta- sheet, ordered, 10-nm diameter, origin-amylose), inclusion bodies (in vivo disordered aggregates, 1  m), disordered aggregates (in vitro) Amyloid fibril formation ubiquitous in polypeptides in nonnative conditions 6

7 * † † Role of Intermediates *disassemble in slightly non-native conditions; † dissassemble in highly non-native conditions

Amyloid Fibrils nm 6 nm

What is wrong with aggregation? Aggregation leads to reduced biological activity; more than 20 protein aggregation diseases (Alzheimer’s, Parkinson’s, mad-cow, ALS,…) each associated with a different protein (  -protein,  -synuclein, prion,…) Insoluable aggregates cause problems in manufacture of protein pharmaceuticals and X-ray crystallography and other structural studies 9

(a) Intranuclear (INI) and cytoplasmic inclusions (CI) in motor cortex of Huntington's disease brain recognized with 1C2 antibody. (b) Lewy body (LB) and other cytoplasmic inclusions (CI) that contain alpha-synuclein within a neuron of the substantia nigra of Parkinson's disease brain. (c) Neuritic plaque of Alzheimer's disease in cerebral cortex. Hirano silver stain identifies intracellular and extracellular protein aggregates. (d) Intranuclear inclusion in frontal cortex of Huntington's disease brain recognized with anti-ubiquitin antibody. (e) Neurofibrillary tangles of Alzheimer's disease in hippocampus immunostained with antibody specific for phosphorylated tau. (f) Diffuse plaque of Alzheimer's disease in cerebral cortex. Amyloid beta (Abeta)-specific antibody recognizes extracellular deposits of Abeta (surrounding a neuron (N) and a capillary (C)). Plaques and Inclusions in Diseased Brains 10

Open Questions What protein species (partially folded, unfolded) is responsible for aggregation? What is the structure of aggregates? What are the physical mechanisms and kinetics that lead to aggregation? Why are some aggregates ordered and others not? 11

Computational Approaches “The competition between protein folding and aggregation: Off- lattice minimalist model studies,” Biotechnology & Bioengineering 89 (2005) 78. B 9 N 3 (LB) 4 N 3 B 9 N 3 (LB) 5 L B=hydrophobic (black) L=hydrophilic (white) N=neutral native state N=neutral (gray) 12

PBC’s BB=10 -4 BB=1 Numerical Study of Aggregation

14 3. trimers 2. one dimer; one folded monomer Outcomes 1. Three folded monomers

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References “Protein aggregation: folding aggregates, inclusion bodies and amyloid,” Folding & Design 3 (1998) R9. “Protein misfolding, evolution and disease,” Trends in the Biochemical Sciences 24 (1999) 329. “Molecular simulation of protein aggregation,” Biotechnology & Bioengineering 96 (2007) 1. “Coarse-grained models for proteins,” Current Opinion in Structural Biology 15 (2005) 144. “Protein-folding landscapes in multichain systems, Proceedings of the National Academy of Sciences 102 (2005) “The competition between protein folding and aggregation: Off- lattice minimalist model studies,” Biotechnology & Bioengineering 89 (2005)