Protein folding @ sid

PROTEIN FOLDING &PROTEIN FOLDING &
RAMACHANDRAN PLOTRAMACHANDRAN PLOT
Siddhartha Swarup Jena
RAD/10-30
Ph.D. Mol. Bio & Biotech
Siddhartha Swarup Jena
RAD/10-30
Ph.D. Mol. Bio & Biotech

What is protein folding?What is protein folding?
 Proteins are amino acid chains that acquire theirProteins are amino acid chains that acquire their
biological and biochemical properties by foldingbiological and biochemical properties by folding
into unique 3-dimensional structures.into unique 3-dimensional structures.
 The shape into which a protein naturally folds isThe shape into which a protein naturally folds is
known as itsknown as its native statenative state, which is, in most cases,, which is, in most cases,
determined only by its sequence of amino acids.determined only by its sequence of amino acids.
 Protein folding is commonly a fast or very fastProtein folding is commonly a fast or very fast
process taking no more than aprocess taking no more than a few millisecondsfew milliseconds
to occur.to occur. S S JenaS S Jena

Folding depends only on primary structureFolding depends only on primary structure
 In 1954In 1954 Christian AnfinsenChristian Anfinsen demonstrated that thedemonstrated that the
folding of a protein in a given environment dependsfolding of a protein in a given environment depends
only on its primary structure - the amino acidonly on its primary structure - the amino acid
sequence.sequence.
 Protein folding is aProtein folding is a thermodynamically driventhermodynamically driven
process: that is, proteins fold by reaching theirprocess: that is, proteins fold by reaching their
thermodynamically most stable structure.thermodynamically most stable structure.
 Hydrophobic amino acidsHydrophobic amino acids will tend to be keptwill tend to be kept insideinside
the structure, with little or no contact with thethe structure, with little or no contact with the
surrounding water; conversely,surrounding water; conversely, polar or chargedpolar or charged
amino acidsamino acids will be oftenwill be often exposedexposed to solvent.to solvent.
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Physiological proteins exist in the folded or “native” state,Physiological proteins exist in the folded or “native” state,
the state with the lowest free energythe state with the lowest free energy
Proteins unfold into a “random coil” if temperature raised orProteins unfold into a “random coil” if temperature raised or
denaturant (urea, GuHCl) addeddenaturant (urea, GuHCl) added
UnfoldedUnfolded
(high T or high denaturant)(high T or high denaturant)
FoldedFolded
(moderate T or low denaturant)(moderate T or low denaturant)

Intramolecular forces in protein foldingIntramolecular forces in protein folding
 The tertiary structure is held together byThe tertiary structure is held together by
hydrogen bonds,hydrogen bonds,
hydrophobic interactions,hydrophobic interactions,
ionic interactions, and/orionic interactions, and/or
disulfide bonds.disulfide bonds.
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Primary structurePrimary structure
 Primary structure is practically a synonym of thePrimary structure is practically a synonym of the
amino acid sequence.amino acid sequence.
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Common Secondary Structure ElementsCommon Secondary Structure Elements
 The Alpha HelixThe Alpha Helix
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Model ofModel of
αα-helix-helix
H bonds
CO HN
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αα -helical coiled coils-helical coiled coils
 2 or more2 or more αα-helices entwined - very strong-helices entwined - very strong
 e.g. Myosin, tropomyosin in muscle,e.g. Myosin, tropomyosin in muscle,
Fibrin in blood clots, Keratin in hairFibrin in blood clots, Keratin in hair
SUPERHELIX S S JenaS S Jena

Common Secondary Structure ElementsCommon Secondary Structure Elements
The Beta SheetThe Beta Sheet
 H bondingH bonding betweenbetween
CO and NH onCO and NH on
differentdifferent
polypeptide strandspolypeptide strands
 Strands can run inStrands can run in
same directionsame direction
((parallelparallel ββ-sheet-sheet))
or in oppositeor in opposite
direction (direction (anti-anti-
parallelparallel ββ-sheet-sheet))
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Model ofModel of ββ-sheet-sheet
 e.g. SILK - anti-parallele.g. SILK - anti-parallel ββ-sheets-sheets
Anti-parallel
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Parallel
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Polypeptide chainPolypeptide chain
folding in proteinsfolding in proteins
illustrating the right-illustrating the right-
handed twist ofhanded twist of ββ
sheets: bovinesheets: bovine
carboxypeptidase A.carboxypeptidase A.
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Collagen triple helixCollagen triple helix
 (Gly-X-Y-Gly-X-Y-Gly-X-Y……)(Gly-X-Y-Gly-X-Y-Gly-X-Y……)
 Glycine occupies every 3rd positionGlycine occupies every 3rd position
- small, fits in interior- small, fits in interior
 Proline and hydroxyproline to exteriorProline and hydroxyproline to exterior
Each strand forms a helix.
3 collagen strands entwine - triple helix
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Collagen triple helixCollagen triple helix
X-Ray structure of the triple helical collagen modelX-Ray structure of the triple helical collagen model
peptide (Pro-Hyp-Gly)peptide (Pro-Hyp-Gly)1010..
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Super secondary StructureSuper secondary Structure
 Proteins can form complicated superstructuresProteins can form complicated superstructures
and patterns of secondary structure elements asand patterns of secondary structure elements as
they fold.they fold.
 Refers toRefers to
elements ofelements of
folding that, dofolding that, do
not neatly fit intonot neatly fit into
the category ofthe category of
tertiary structure.tertiary structure.
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Super secondary StructureSuper secondary Structure
Schematic diagrams of super-secondary structures.Schematic diagrams of super-secondary structures.
βαβ β-
hairpin
αα
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Secondary Structure & Protein FoldingSecondary Structure & Protein Folding
 Hydrophobicity is a large factor that determinesHydrophobicity is a large factor that determines
the final folded shape of the proteinthe final folded shape of the protein
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Tertiary StructureTertiary Structure
Lactate
Dehydrogenase:
Mixed α / β
Immunoglobu
lin Fold: β
Hemoglobin B,
Chain: α
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TertiaryTertiary
StructureStructure
bondsbonds
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Quaternary StructureQuaternary Structure
 Complex of 2 or more separate polypeptideComplex of 2 or more separate polypeptide
chainschains
 Interactions between subunitsInteractions between subunits
 Non-covalent and covalent interactionsNon-covalent and covalent interactions
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Model of hemoglobinModel of hemoglobin
 4 subunits4 subunits
interactinginteracting
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Representations of the X-ray structure ofRepresentations of the X-ray structure of
sperm whale myoglobinsperm whale myoglobin
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Summary of protein structureSummary of protein structure
1Y
2Y
3Y
4Y
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Ramachandran PlotRamachandran Plot
 The Ramachandran plot is aThe Ramachandran plot is a
fundamental tool in thefundamental tool in the analysis ofanalysis of
protein structuresprotein structures..
 Invented by professorInvented by professor G. N.G. N.
RamachandranRamachandran, a very eminent scientist, a very eminent scientist
from India.from India.
Prof. G. N.
Ramachandran
 A graphical representation in which the dihedral angleA graphical representation in which the dihedral angle
of rotation about theof rotation about the alpha-carbon to carbonyl-carbonalpha-carbon to carbonyl-carbon
bondbond in polypeptides is plotted against the dihedralin polypeptides is plotted against the dihedral
angle of rotation about theangle of rotation about the alpha-carbon to nitrogenalpha-carbon to nitrogen
bondbond..
 He also discovered the triple helix structure ofHe also discovered the triple helix structure of
collagen in 1954.collagen in 1954. S S JenaS S Jena

 G N Ramachandran usedG N Ramachandran used
computer models ofcomputer models of
small polypeptides tosmall polypeptides to
systematically vary phisystematically vary phi
and psi with the objectiveand psi with the objective
of finding stableof finding stable
conformations.conformations.
 In a polypeptide the main chainIn a polypeptide the main chain N-CN-Cαα andand CCαα-C-C
bonds are relatively free to rotate. These rotationsbonds are relatively free to rotate. These rotations
are represented by the torsion anglesare represented by the torsion angles phi (phi (ΦΦ)) andand
psi (psi (ψψ)), respectively., respectively.
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Phi & Psi AnglesPhi & Psi Angles
 Rotational constraints emerge from interactionsRotational constraints emerge from interactions
with bulky groups (i.e. side chains).with bulky groups (i.e. side chains).
 Phi & Psi angles define the secondary structurePhi & Psi angles define the secondary structure
adopted by a protein.adopted by a protein.
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Allowed phi and psi torsion anglesAllowed phi and psi torsion angles
in proteins.in proteins.
The Ramachandran diagramThe Ramachandran diagram
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Protein Structure DatabasesProtein Structure Databases
 PDBPDB:: (Protein Data Bank)(Protein Data Bank)
 http://www.rcsb.org/pdb/http://www.rcsb.org/pdb/
 MMDBMMDB:: (Molecular Modeling Database)(Molecular Modeling Database)
 http://www.ncbi.nlm.nih.gov/Structure/http://www.ncbi.nlm.nih.gov/Structure/
 PQS:PQS: ((protein quaternary structure query formprotein quaternary structure query form))
 http://pqs.ebi.ac.ukhttp://pqs.ebi.ac.uk
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Protein Structural classification algorithmProtein Structural classification algorithm
 FSSPFSSP:: (Fold classification based on structure-structure(Fold classification based on structure-structure
alignment of proteins)alignment of proteins)
 http://www.ebi.ac.uk/dali/fssp/http://www.ebi.ac.uk/dali/fssp/
 SCOPSCOP:: (Structural Classification of Proteins)(Structural Classification of Proteins)
 http://scop.mrc-lmb.cam.ac.uk/scop/http://scop.mrc-lmb.cam.ac.uk/scop/
 CATHCATH:: [Class(C), Architecture(A), Topology(T) and[Class(C), Architecture(A), Topology(T) and
Homologous (H) super family]Homologous (H) super family]
 http://www.biochem.ucl.ac.uk/bsm/cath_newhttp://www.biochem.ucl.ac.uk/bsm/cath_new//
 CE:CE: ((combinatorial extension of optical pathwaycombinatorial extension of optical pathway))
 http://cl.sdsc.eduhttp://cl.sdsc.edu
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Levinthal paradoxLevinthal paradox
 The protein folding problem relates to what isThe protein folding problem relates to what is
known as theknown as the Levinthal paradoxLevinthal paradox..
 If a fairly small protein is composed ofIf a fairly small protein is composed of 100 amino100 amino
acidsacids and each amino acid residue has only 3and each amino acid residue has only 3
possible conformations then the entire protein canpossible conformations then the entire protein can
fold intofold into 33100100
-1-1 or 5x10or 5x104747
possible conformations.possible conformations.
 Even if it takes onlyEven if it takes only 1010-13-13
of a secondof a second to try eachto try each
conformation it would takeconformation it would take 10102727
yearsyears to try them all.to try them all.
 Obviously a protein doesn't take that long to fold, soObviously a protein doesn't take that long to fold, so
protein folding is not random, must have pathwaysprotein folding is not random, must have pathways..
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The protein folding problemThe protein folding problem
 The protein folding problem which hasThe protein folding problem which has
perplexed scientists for over thirty years is thatperplexed scientists for over thirty years is that
of understanding how the tertiary structure of aof understanding how the tertiary structure of a
protein is related to its primary structure,protein is related to its primary structure,
because it has been proven that thebecause it has been proven that the primaryprimary
structure of a protein holds the only informationstructure of a protein holds the only information
necessary for the protein to foldnecessary for the protein to fold..
 Ultimately the aim is also to be able to predictUltimately the aim is also to be able to predict
what pathway the protein will take.what pathway the protein will take.
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Anfinsen ExperimentAnfinsen Experiment
 Denaturation ofDenaturation of
ribunuclease A ( 4ribunuclease A ( 4
disulfide bonds) withdisulfide bonds) with
8 M Urea8 M Urea
containingcontaining ββ--
mercaptoethanol tomercaptoethanol to
random coil,random coil,
 No activity foundNo activity found
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Anfinsen ExperimentAnfinsen Experiment
 After renaturation, the refolded protein has nativeAfter renaturation, the refolded protein has native
activity despite the fact that there are 105 ways toactivity despite the fact that there are 105 ways to
renature the protein.renature the protein.
 Conclusion:Conclusion: All the information necessary for foldingAll the information necessary for folding
the peptide chain into its native structure is containedthe peptide chain into its native structure is contained
in the primary amino acid sequence of the peptide.in the primary amino acid sequence of the peptide.
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Protein folding as a reactionProtein folding as a reaction
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Models for Protein FoldingModels for Protein Folding
 Framework model: (Ptittsyn)Framework model: (Ptittsyn)
 Folding is thought to start with the formation of secondaryFolding is thought to start with the formation of secondary
structure independently of tertiary structure. Thenstructure independently of tertiary structure. Then
assemble into tightly packed native tertiary structure.assemble into tightly packed native tertiary structure.
 Hydrophobic collapse model (Dill)Hydrophobic collapse model (Dill)
 Initial event of the reaction is thought to be a relativelyInitial event of the reaction is thought to be a relatively
uniform collapse of the protein molecule driven byuniform collapse of the protein molecule driven by
hydrophobic effect.hydrophobic effect.
 Nucleation-condensation mechanism (Fersht)Nucleation-condensation mechanism (Fersht)
 Early formation of a diffuse protein folding nucleusEarly formation of a diffuse protein folding nucleus
catalyzes further folding. The nucleus primarily consistscatalyzes further folding. The nucleus primarily consists
of a few adjacent residues which have some correctof a few adjacent residues which have some correct
secondary structure interaction.secondary structure interaction.
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Models for Protein FoldingModels for Protein Folding
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The Folding FunnelThe Folding Funnel
 Progress from the topProgress from the top
to the bottom of theto the bottom of the
funnel isfunnel is
accompanied by anaccompanied by an
increase in theincrease in the
native-like structurenative-like structure
as folding proceeds.as folding proceeds.
 A new view of protein folding suggested that there isA new view of protein folding suggested that there is
no single route, but a large ensemble of structuresno single route, but a large ensemble of structures
follow a many dimensional funnel to its nativefollow a many dimensional funnel to its native
structure.structure.
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Protein Disulfide Isomerase (PDI)Protein Disulfide Isomerase (PDI)
 The formation of correctThe formation of correct
disulfide pairings in nascentdisulfide pairings in nascent
proteins is catalyzed by PDI.proteins is catalyzed by PDI.
 PDI preferentially binds withPDI preferentially binds with
peptides that contain Cyspeptides that contain Cys
residues.residues.
 By shuffling disulfide bonds,By shuffling disulfide bonds,
PDI enables proteins toPDI enables proteins to
quickly find thequickly find the
thermodynamically mostthermodynamically most
stable pairing that arestable pairing that are
accessible.accessible.
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Peptidyl Prolyl Isomerase (PPI)Peptidyl Prolyl Isomerase (PPI)
 Prolyl isomerization is theProlyl isomerization is the
rate-limiting in the folding ofrate-limiting in the folding of
many proteins in vitro.many proteins in vitro.
 PPI accelerates cis-transPPI accelerates cis-trans
isomerization more than 300isomerization more than 300
fold by twisting the peptidefold by twisting the peptide
bond so that the C,O, and Nbond so that the C,O, and N
atoms are no longer planar.atoms are no longer planar.
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Molecular ChaperonesMolecular Chaperones
 Nascent polypeptides come off the ribosome and foldNascent polypeptides come off the ribosome and fold
spontaneously; molecular chaperones are involved inspontaneously; molecular chaperones are involved in
their folding in vivo, and are related to heat shocktheir folding in vivo, and are related to heat shock
proteins (hsp).proteins (hsp).
 The main hsp families are:The main hsp families are:
 "Small hsp's" - Diverse "family" 10,000 - 30,000 MW"Small hsp's" - Diverse "family" 10,000 - 30,000 MW
(hsp26/27 - crystallins (eye lens))(hsp26/27 - crystallins (eye lens))
 hsp40hsp40
 hsp60 (e.g. GroEL in E. coli)hsp60 (e.g. GroEL in E. coli)
 hsp70 (DnaK in E. coli)hsp70 (DnaK in E. coli)
 hsp90hsp90
 hsp100hsp100
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Folding in extreme environmentsFolding in extreme environments
 Most proteins are not capable of maintaining their three-Most proteins are not capable of maintaining their three-
dimensional shape when exposed to environmentaldimensional shape when exposed to environmental
extremes such as aextremes such as a low or high pHlow or high pH, or a, or a highly variablehighly variable
temperaturetemperature..
 Changes in the pH of the proteins environment may alterChanges in the pH of the proteins environment may alter
the charges on the amino acid side chains, altering thethe charges on the amino acid side chains, altering the
secondary and tertiary structure of the protein as asecondary and tertiary structure of the protein as a
whole, as a result the shape of the enzyme is warped.whole, as a result the shape of the enzyme is warped.
 Certain proteins, mainly digestive enzymes suchCertain proteins, mainly digestive enzymes such
asas trypsintrypsin, are capable of with-standing a, are capable of with-standing a pH as low aspH as low as
11. If the pH of such an enzymes environment were to. If the pH of such an enzymes environment were to
increase to approximately pH 5, it would be inactivated.increase to approximately pH 5, it would be inactivated.
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Importance of Protein FoldingImportance of Protein Folding
 Avoid misfolding related to human diseasesAvoid misfolding related to human diseases
 Design proteins with novel functionsDesign proteins with novel functions
 3-Dimensional structure useful in molecular3-Dimensional structure useful in molecular
drug design.drug design.
 Genome projects are providing sequences forGenome projects are providing sequences for
many proteins whose structure will need to bemany proteins whose structure will need to be
determined.determined.
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Protein misfoldingProtein misfolding
 If the protein misfolds, its properties can beIf the protein misfolds, its properties can be
markedly changed, as the way protein foldsmarkedly changed, as the way protein folds
determines which active groups are exposeddetermines which active groups are exposed
for interaction.for interaction.
 One example of this is in TransmissibleOne example of this is in Transmissible
Spongiform Encephalopathies, such asSpongiform Encephalopathies, such as BSEBSE,,
andand ScrapieScrapie..
 In these, theIn these, the prion proteinprion protein, which is involved in, which is involved in
thethe brain'sbrain's copper metabolismcopper metabolism, misfolds, and, misfolds, and
starts forming plaques, which destroy brainstarts forming plaques, which destroy brain
tissue.tissue.
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Protein misfoldingProtein misfolding
Disease Protein misfolded
Pick’s
Alzheimer’s
Parkinson’s
Prion disease (e.g. Mad Cow)
Amyloid Lateral Sclerosis
( Lou Gehrig’s)
Huntington’s Disease
tau
A-beta
alpha synuclein
prion protein
TDP-43
Huntingtin
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Protein folding @ sid

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