2026 Intro fall2018 - University of Toronto
Transcript of 2026 Intro fall2018 - University of Toronto
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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 [email protected], [email protected]
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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