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JBB 2026H: Protein Structure, Folding and Design

University of TorontoRecommended for students interested in understanding more

about structural biology and biophysics to better understand biological mechanisms and function

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 2018 Fridays 10:00 am -- 12:00

noon Medical Sciences Building (MSB), Room 3278Additional 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%)1

Recent studies have shown how a variety of different protein conformational states, including folded, dynamic, disordered, amyloid, gel, etc., can serve biological function or cause diseases.

Thus, in addition to the more standard emphasis on folded and unfolded structures, we provide necessary physical concepts for students to understand the multiplicity of conformational states available to proteins as well as examples of the functional relevance of these states.

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Overview: The Continuum of Protein Structural States

Biochemistry - chemistry of biological systems/lifeProteins, nucleic acids, lipids, carbohydrates, small molecules, waterPhysical 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 signalingbased on protein liquid-liquid demixing 3

B. Importance of disorder/motion/dynamics along with orderneed organization, ordered chemistry to have efficient catalysis, ordered complexes for efficient and specific biological processes

But, life is not static – responsive dynamic processesneed to facilitate dynamics in molecules of lifebond making/breaking, synthesis/degradation of biomoleculesdynamics 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

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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

Proteinpolymer

Students are expected to know the 20 aa residue sidechains and their chemical properties (charge, aromaticity, polarity, size, hydrophobicity, etc) 5

Proteins are specialized example of an organic polymer - used primarily for synthetic materials: plastics, nylon (polyamide)

poly(oxyethylene)

(CH2 CH2 O)N

polystyrene

(CH2 C H)Nmainchainpolymers

sidechainpolymers

branchedchainpolymers

Hydrogen bonding in nylon 6,6 parallel b-sheets (rt)differ from protein in that proteins only

have one carbon atom between amide groupsother nylons have different #s of carbons and

can form parallel & anti-parallel sheetsthermoplastics (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 proteinsusually proteins phase separate at UCST

solvated phase separated7

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Proteins are a unique polymer capable of specific ordered folding or fluctuating structure or highly diverse conformational equilibrium

Structural propensities are defined by torsion angle distributions(torsion angle rotation for covalently bonded atoms A-B-C-D defined as the angle between the two planes A-B-C and B-C-D)Phi: CO(i-1)-N-Ca-CO Psi: N-Ca-CO-N(i+1)Omega: Ca-CO-N(i+1)-Ca(i+1) is always very close to either trans (mostly) or cis (rarely, X-Pro peptide bonds) due to the peptide double bond character and planarity, so protein structure can be largely understood as phi,psi distributions

Folded proteins have discrete phi,psi torsion angles for each residue with minimal fluctuationsDisordered proteins have large distributions of phi,psi torsion angles

Ability to use polymer theory (HS Chan)- statistical chain models with conformational entropy, …

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Proteinsmore 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 dotuned more finely, variability in propertiestransparancy, tensile/shear strength, elasticity, temp dependence

Can do more than organic polymersLarge 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:(i) proteins are polymers with a very large potential for

conformational alternatives, and (ii) evolution/nature has made functional use of all possible states 9

Protein states Virtually all properties are on a continuumwe 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

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Monomers(1) 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

Higher Order Assemblies(1) stable fibrils, amyloid, b-aggregates (functional/pathological)(2) gels, elastic protein aggregates (3) disordered liquid/dynamic aggregates

liquid-liquid phase separated aggregated states of disordered/multi-valent/dynamic proteins

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proteinsynthesis

folded

foldingintermediate

Protein states - monomers

unfolded

intrinsicallydisordered

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Protein states - oligomers

20Sproteasome

DiscreteIntermediateinAggregation

Molecularmachinery

ATPsynthase*

Multimericproteinassembly

Discretedisorderedproteinassociation–dynamiccomplex

Dynamic/fuzzycomplexes

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Maynotalwaysbediscrete….

Protein states - higher order assembliesMesoscale/MacroscaleAssemblies

Nonfunctionalaggregates

AmyloidFibrils

LiquidassemblyNon-membrane-boundorganelles

ElastinGelassembly

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DefinitionsFolded state:

more ordered state comprised of a fairly tight distribution ofconformations near the lowest energy structure

Disordered state: ensemble of rapidly interconverting conformers multiple structures thought to have similar energiesgeneral 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.

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Random coilrandom 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 residuesvery unstable under physiological aqueous conditionsmay be approached under highly denaturing conditionssome conformations extended, some compact

Statistical coilmodification of concept of random coilsampling 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 statehigher energy state lacking folded structural stabilization yet

present under non-denaturing conditions16

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Intermediate stateenergy higher than folded stateensemble of interconverting conformers rapidly fluctuating 2° and 3° structural contacts

Molten globule (MG) - canonical descriptionradius of gyration between folded and denatured state2° structure content high (CD, NMR), 3° structure low/transientsidechain environment water accessibleenthalpy change associated with F <--> U

occurs in F <--> MG, not in MG <--> U step

Smith et al, Folding & Design 1:R95-R106, 1996.

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Native statesignificantly populated under native physiological conditions, often the folded state although not alwaysincorrectly used as a synonym for folded stateintrinsically 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 functionalnumerous intrinsically disordered proteins have been described, involved in recognition, “polymer properties”more disorder is predicted for more complex organisms and

regulatory proteinspredict % of proteins with stretches of >= 30 disordered residues

eukaryotic – 33%; bacterial – 4%; signaling/cancer-associated – 50-80%

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Intrinsically disordered proteins (cont)** primary role in mediating protein recognition and association

disorder-to-order transitions can be local or globalcomplete, partial or very minimal/transient (dynamic complex)

entropic penalty balanced with often extended bindinginterface 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

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Dynamic complex / fuzzy complexpartial disorder-to-order transition of IDP upon binding to targetdynamic exchange between multiple discrete interactions

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Dynamic / fuzzy complex (example)Cdc4 (subunit of ubiquitin ligase) interacting with disordered Sic1 (cyclin dependent kinase inhibitor) via exchanging multivalent interactions of phosphorylated motifs in an IDP with binding surfaces on a folded domain

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Mittag et al, PNAS 2008Borg et al, PNAS 2007Mittag et al, Structure 2010Csizmok et al, Nature Commun 2017 22

Disordered complex (two disordered proteins binding)histone H1 (+53) and its nuclear chaperone prothymosin-α (-44) associate in a complex with picomolar affinity, but fully retain their structural disorder and long-range flexibility- dominant role of electrostatic

interactions

A Borgia, …, R Best, B Schuler, Nature 2018

Fibersstable folded cores with specific oligomerization but large Ncollagen, actin, other cytoskeletal proteins

Amyloidvery stable aggregate with high beta-sheet contentbinds Congo red & other dyes, typically cross-beta structure often self-templating growthcan 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 pathologicalamyloidosesPrion protein, abetapossibly infectious

FibrilAxisPetkovaetal.(2006)Biochemistry 23

Gel, hydrogelhydrated, 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 separationhydrated and strongly self-associated protein in a separate “phase” from solvent, phase transition dependent on conditions

very dynamic sampling of multiple, weak interactionscan be heterotypic (ie multi-valent SH3 domain/Pro-rich motif)

lead to (often spherical) “organelles” or protein bodiesexamples are nucleoli, P bodies, actin-polymerizing puncta

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