JBB 2026H: Protein Structure, Folding and Design University of Toronto

1. JBB 2026H: Protein Structure, Folding and Design University of Toronto Recommended for trainees of the CIHR Strategic Training Program in Protein Folding and Interaction Dynamics Coordinators: Hue Sun Chan and Julie Forman-Kay chan@arrhenius.med.utoronto.ca, forman@sickkids.ca Emphasizes basic biophysical concepts Lectures: Sept 14 to Dec 7 2012 Fridays 10:00 am -- 12:00 noon Medical Sciences Building (MSB), Room 3163 Additional sessions will be scheduled for student presentations before and/or soon after the final examination. Evaluation: Student presentation (25%); assignments (25%); participation (10%); final exam (40%)

2. Overview: The Continuum of Protein Structural StatesBiochemistry - chemistry of biological systems/life • Proteins, nucleic acids, lipids, carbohydrates, small molecules, water • Physical chemistry of biomolecules is exploited to enable biology • forces: electrostatics, hydrophobic interactions, vdW,etc • ** energy landscapes of single molecules (intramol contacts) • ** energetics of association (intermolecular contacts) • as function of conditions pH/temp/salt/small molecules • What makes chemistry of life different? • A. Organization • cells surrounded by lipid bilayer, inside is water, compartments • - lipid bilayers (nuclei, mitochondria, lysozomes, endosomes, etc) • non-membrane bound organelles (RNA processing, nucleoli, etc) • these are much more dynamic, responsive to signaling • based on protein liquid-liquid demixing

3. fjdskllllllllllllllllllllll B. Importance of disorder/motion/dynamics along with order need organization, ordered chemistry to have efficient catalysis, ordered complexes for efficient and specific biological processes But, life is not static – responsive dynamic processes need to facilitate dynamics in molecules of life bond making/breaking, synthesis/degradation of biomolecules dynamics in conformational sampling of biomolecules is key enzyme catalysis, organization, responsiveness C. Evolutionary tuning of chemistry via protein sequences primarily indirectly for other biochemicals via evolution of enzymes variety of functional protein states are targets of evolution possibility that any of these could be pathological (ie a particular physical state itself is not pathological)

4. Proteins as polymersproteins are linear polymers of amino acids, typically the 20 residues types utilized in cellular protein synthesis by the ribosome by translating a three-nucleotide code with specific acyl-aa tRNAs Protein polymer Students expected to know the 20 aa residue sidechains and their chemical properties (charge, aromaticity, polarity, size, hydrophobicity, etc)

5. Proteins are specialized example of other organic polymers - used primarily for synthetic materials: plastics, nylon (polyamide) main chain polymers polystyrene poly (oxyethylene) side chain polymers branched chain polymers Hydrogen bonding in nylon 6,6 parallel b-sheets (rt) differ from protein in that proteins only have one carbon atom between amide groups other nylons have different #s of carbons and can form parallel & anti-parallel sheets thermoplastics (nylon) can also be amorphous solids or viscous fluids above melting temps at which chains approach random coil behavior (above)

6. Polymers (cont) Poly-NIPAM (PNIPAM) is a temperature sensitive polymer exhibits hydrophobic-hydrophilic phase transition at its LCST (lower critical solution temperature) evidence that monomers collapse and then aggregate to form phase separated hydrogel used in various applications - drug delivery, filtration similar phase separation as for proteins usually proteins phase separate at UCST solvated phase separated

7. Proteins more heterogeneity in building blocks than organic polymers (>20) highly uniform sequence that can be acted upon by evolution Can do (probably) everything that organic polymers can do tuned more finely, variability in properties transparancy, tensile/shear strength, elasticity, temp dependence Can do more than organic polymers Large variety of types of structures accessible by protein polymers Due to their unique ability to fold into relatively ordered monomeric structures with alpha-helical and beta-sheet structures the rest of the landscape of available conformational space has often been ignored Here we emphasize that: proteins are polymers with a very large potential for conformational alternatives, and evolution/nature has made functional use of all possible states

8. Protein states Virtually all properties are on a continuum we will artifically divide two significant properties into 3 regions N (how many protein molecules): monomers -> dimer/trimer/n-mer/specific homo- and hetero- oligomers -> higher order assemblies (N is large and not specifically defined) Energetics/dynamics: stable, low energy ground state, minimal dynamic excursions -> sampling (discrete) multiple conformations, intermediate dynamics -> disordered, rapid sampling of large number of conformations

9. Monomers stable folded ground states (2) higher energy/more dynamic states sampled from folded state intermediate, molten globule, excited states, partially folded (3) disordered states (unfolded, intrinsically disordered proteins) Dimer/Trimer/Specific Homo and Hetero-Oligomers (1) stable folded oligomeric states homo- or hetero-oligomers, supramolecular complexes (2) dynamic complexes sampling multiple possible interactions (3) disordered complexes (ex. dimers of T cell receptor  chain) Higher Order Assemblies stable fibrils, amyloid, -aggregates (functional/pathological) gels, elastic protein aggregates (3) disordered liquid/dynamic aggregates liquid-liquid phase separated aggregated states of disordered/multi-valent/dynamic proteins

10. Protein states - monomers protein synthesis unfolded folded intrinsically disordered folding intermediate Aggregation/fibrils

11. Protein states - oligomers Multimeric protein assembly Molecular machinery 20S proteasome ATP synthase* Dynamic/fuzzy complexes IntermediateAggregation Disordered protein association

12. Protein states - higher order assembliesMesoscale/Macroscale Assemblies Amyloid Fibrils Elastin Gel assembly Liquid assembly Non-membrane- bound organelles Nonfunctional aggregates

13. Definitions Folded state: more ordered state comprised of a fairly tight distribution of conformations near the lowest energy structure Disordered state: ensemble of rapidly interconverting conformers multiple structures thought to have similar energies general term including random coil, statistical coil, unfolded, denatured, partially folded/unfolded, intrinsically disordered Schematic diagram representing a disordered state, demonstrating an inter-converting ensemble of multiple conformations. The backbone of the chain is colored at various points to aid in visualization of the distinct conformers and residual turn structure.

14. Random coil random interactions between all amino acids (i.e. no specific sidechain-sidechain or non-local interactions) backbone and sidechain torsion angles for a particular residue independent of all other residues very unstable under physiological aqueous conditions may be approached under highly denaturing conditions some conformations extended, some compact Statistical coil modification of concept of random coil sampling range of backbone and sidechain torsion angles expected for particular amino acid type (database) Denatured state generated in the presence of denaturing conditions (i.e. Gdm+Cl- or urea, acid or base, high/low temperature) Unfolded state higher energy state lacking folded structural stabilization yet present under non-denaturing conditions

15. Smith et al, Folding & Design 1:R95-R106, 1996. Intermediate state energy higher than folded state ensemble of interconverting conformers rapidly fluctuating 2° and 3° structural contacts Molten globule (MG) - canonical description radius of gyration between folded and denatured state 2° structure content high (CD, NMR), 3° structure low/transient sidechain environment water accessible enthalpy change associated with F <--> U occurs in F <--> MG, not in MG <--> U step

16. Native state significantly populated under native physiological conditions, often the folded state although not always incorrectly used as a synonym for folded state intrinsically disordered proteins can be disordered under native conditions Intrinsically disordered proteins (and protein regions) - IDP, IDR (natively unfolded, natively disordered, intrinsically unstructured) proteins do not necessarily need to be folded to be functional numerous intrinsically disordered proteins have been described, involved in recognition, “polymer properties” more disorder is predicted for more complex organisms and regulatory proteins predict % of proteins with stretches of >= 30 disordered residues eukaryotic – 33%; bacterial – 4%; signaling/cancer-associated – 50-80%

17. Intrinsically disordered proteins (cont) ** primary role in mediating protein recognition and association disorder-to-order transitions can be local or global complete, partial or very minimal/transient (dynamic complex) entropic penalty balanced with often extended binding interface with large surface area fine tunes thermodynamic/kinetic binding properties contain sites for post-translational modifications and binding to modular binding domains flexible, plastic binding, potential for enhanced binding kinetics ** “polymer” state of protein -> yield unusual higher order states variable material and other properties elastic, proteinaceous detergent - amyloid-like more rigid beta-associated states can undergo liquid-liquid demixing self-association to form - hydrogels - liquid-like non-membrane-bound organelles

18. Dynamic complex / fuzzy complex partial disorder-to-order transition of IDP upon binding to target dynamic exchange between multiple discrete interactions

19. Dynamic complex / fuzzy complex Example - Cdc4 (subunit of ubiquitin ligase) interacting with disordered Sic1 (cyclin dependent kinase inhibitor)

20. Fibers stable folded cores with specific oligomerization but large N collagen, actin, other cytoskeletal proteins Amyloid very stable aggregate with high beta-sheet content binds Congo red & other dyes, typically cross-beta structure often self-templating growth can be functional Heinrich & Lindquist (2011) Protein-only mechanism induces self-perpetuating changes in the activity of neuronal Aplysia cytoplasmic polyadenylation element binding protein (CPEB), PNAS 108, 2999-3004. Majumdar et al (2012) Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory, Cell 148, 515-529. Li et al (2012) The RIP1/RIP3 Necrosome Forms a Functional Amyloid Signaling Complex Required for Programmed Necrosis. Cell 150, 339-50. can be pathological amyloidoses Prion protein, abeta possibly infectious Petkova et al. (2006) Biochemistry Fibril Axis

21. Gel, hydrogel hydrated, strongly self-associated protein dynamic at an intermediate level due to “cross-links” of strong interactions that are semi-specific (beta-sheet type, cation-pi) example is thought to be in nucleoporin pore where disordered chains create filtration barrier by formation of gel Liquid-liquid demixing / liquid phase separation hydrated and strongly self-associated protein in a separate “phase” from solvent, phase transition dependent on conditions very dynamic sampling of multiple, weak interactions can be heterotypic (ie multi-valent SH3 domain/Pro-rich motif) lead to (often spherical) “organelles” or protein bodies examples are nucleoli, P bodies, actin-polymerizing puncta

22. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes C.P. Brangwynnea, T.J. Mitchison, A.A. Hymana PNAS 108, 4334–9, 2011 Fluid-like behavior of nucleoli. (A) DIC image sequence showing fusion of two spherical nucleoli into one larger spherical nucleolus. (B) DIC image of the fusion of three spherical nucleoli into one larger spherical nucleolus. (C) Plot of the sum of nucleoli volumes before and after fusion. The red line corresponds to conserved volume. (D) In the first frame, three nucleoli that have come into contact and begun fusing are visible. The bridge between the two on the Left is unstable and pinches off, whereas the bridge between the nucleoli on the Right is stable and they fuse. (E) Close-up of the rupturing bridge from D, showing that the threads of nucleolar material are resorbed within 1 min after rupturing.

23. Protein states in the cell functional and pathological Folded Disordered Vendruscolo et al, Cold Spring Habor Perspectives, 2011