PROTEIN FOLDING & PROTEIN FOLDING & RAMACHANDRAN PLOTRAMACHANDRAN PLOT

Siddhartha Swarup JenaRAD/10-30

Ph.D. Mol. Bio & Biotech

Siddhartha Swarup JenaRAD/10-30

Ph.D. Mol. Bio & Biotech

What is protein folding?What is protein folding?

Proteins are amino acid chains that acquire their Proteins are amino acid chains that acquire their biological and biochemical properties by folding biological and biochemical properties by folding into unique 3-dimensional structures. into unique 3-dimensional structures. 

The shape into which a protein naturally folds is The shape into which a protein naturally folds is known as its known 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 fast Protein folding is commonly a fast or very fast process taking no more than a process taking no more than a few milliseconds few milliseconds to occur.to occur.

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Folding depends only on primary structureFolding depends only on primary structure

In 1954 In 1954 Christian AnfinsenChristian Anfinsen demonstrated that the  demonstrated that the folding of a protein in a given environment depends folding of a protein in a given environment depends only on its primary structure - the amino acid only on its primary structure - the amino acid sequence.sequence.

Protein folding is a Protein folding is a thermodynamically driven thermodynamically driven process: that is, proteins fold by reaching their process: that is, proteins fold by reaching their thermodynamically most stable structure.thermodynamically most stable structure.

Hydrophobic amino acids Hydrophobic amino acids will tend to be kept will tend to be kept insideinside the structure, with little or no contact with the the structure, with little or no contact with the surrounding water; conversely, surrounding water; conversely, polar or charged polar or charged amino acidsamino acids will be often will 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 or Proteins 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 folding Intramolecular forces in protein folding

The tertiary structure is held together by The tertiary structure is held together by

hydrogen bonds, hydrogen bonds, hydrophobic interactions, hydrophobic interactions, ionic interactions, and/or ionic interactions, and/or disulfide bonds.disulfide bonds.

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Primary structurePrimary structure

Primary structure is practically a synonym of the Primary 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 of Model of αα-helix-helix

H bondsCO HN

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-helical coiled coils-helical coiled coils

2 or more 2 or more -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

SUPERHELIXS S JenaS S Jena

Common Secondary Structure ElementsCommon Secondary Structure Elements

The Beta SheetThe Beta Sheet

H bonding H bonding between between CO and NH on CO and NH on different different polypeptide strandspolypeptide strands

Strands can run in Strands can run in same direction same direction ((parallel parallel ββ-sheet-sheet))

or in opposite or in opposite direction (direction (anti-anti-parallel parallel ββ-sheet-sheet))

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Model of Model of -sheet-sheet

e.g. SILK - anti-parallel e.g. SILK - anti-parallel -sheets-sheetsAnti-parallel

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Model of Model of -sheet-sheet

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Model of Model of -sheet-sheet

Polypeptide chain Polypeptide chain folding in proteins folding in proteins

illustrating the right-illustrating the right-handed twist of handed twist of sheets: bovine sheets: 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 position Glycine 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 model X-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 superstructures Proteins can form complicated superstructures and patterns of secondary structure elements as and patterns of secondary structure elements as they fold.they fold.

Refers to Refers to elements of elements of folding that, do folding that, do not neatly fit into not neatly fit into the category of the 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.

-hairpin

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Secondary Structure & Protein FoldingSecondary Structure & Protein Folding

Hydrophobicity is a large factor that determines Hydrophobicity 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 /

Immunoglobulin Fold:

Hemoglobin B, Chain:

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Tertiary Tertiary StructureStructure

bondsbonds

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Quaternary StructureQuaternary Structure

Complex of 2 or more separate polypeptide Complex 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 of Representations 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 a The Ramachandran plot is a fundamental tool in the fundamental tool in the analysis of analysis of protein structuresprotein structures. .

Invented by professor Invented 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 angle A graphical representation in which the dihedral angle of rotation about the of rotation about the alpha-carbon to carbonyl-carbon alpha-carbon to carbonyl-carbon bond bond in polypeptides is plotted against the dihedral in polypeptides is plotted against the dihedral angle of rotation about the angle of rotation about the alpha-carbon to nitrogen alpha-carbon to nitrogen bondbond. .

He also discovered the triple helix structure of He also discovered the triple helix structure of collagen in 1954.collagen in 1954.

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Ramachandran PlotRamachandran Plot

G N Ramachandran used G N Ramachandran used computer models of computer models of small polypeptides to small polypeptides to systematically vary phi systematically vary phi and psi with the objective and psi with the objective of finding stable of finding stable conformations.conformations.

In a polypeptide the main chain In a polypeptide the main chain N-CN-Cαα and and CCαα-C -C bonds are relatively free to rotate. These rotations bonds are relatively free to rotate. These rotations are represented by the torsion angles are represented by the torsion angles phi (phi (ΦΦ)) and and psi (psi (ψψ)), respectively., respectively.

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Phi & Psi AnglesPhi & Psi Angles

Rotational constraints emerge from interactions Rotational constraints emerge from interactions with bulky groups (i.e. side chains).with bulky groups (i.e. side chains).

Phi & Psi angles define the secondary structure Phi & Psi angles define the secondary structure adopted by a protein.adopted by a protein.

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Ramachandran PlotRamachandran Plot

Allowed phi and psi torsion angles Allowed phi and psi torsion angles in proteins.in proteins.

The Ramachandran diagramThe Ramachandran diagram

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Ramachandran PlotRamachandran Plot

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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))

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Levinthal paradoxLevinthal paradox

The protein folding problem relates to what is The protein folding problem relates to what is known as the known as the Levinthal paradoxLevinthal paradox..

If a fairly small protein is composed of If a fairly small protein is composed of 100 amino 100 amino acidsacids and each amino acid residue has only 3 and each amino acid residue has only 3 possible conformations then the entire protein can possible conformations then the entire protein can fold into fold into 33100100-1-1 or 5x10 or 5x104747 possible conformations.  possible conformations.

Even if it takes only Even if it takes only 1010-13-13 of a second  of a second to try each to try each conformation it would take conformation it would take 10102727 years  years to try them all.to try them all.

Obviously a protein doesn't take that long to fold, so Obviously 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 has The protein folding problem which has perplexed scientists for over thirty years is that perplexed scientists for over thirty years is that of understanding how the tertiary structure of a of 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 the because it has been proven that the primary primary structure of a protein holds the only information structure 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 predict Ultimately 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 Experiment Anfinsen Experiment

Denaturation of Denaturation of ribunuclease A ( 4 ribunuclease A ( 4 disulfide bonds) with disulfide bonds) with 8 M Urea 8 M Urea containingcontaining--mercaptoethanol to mercaptoethanol to random coil, random coil,

No activity foundNo activity found

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Anfinsen Experiment Anfinsen Experiment

After renaturation, the refolded protein has native After renaturation, the refolded protein has native activity despite the fact that there are 105 ways to activity despite the fact that there are 105 ways to renature the protein.renature the protein.

Conclusion:Conclusion: All the information necessary for folding All the information necessary for folding the peptide chain into its native structure is contained the 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 secondary Folding is thought to start with the formation of secondary

structure independently of tertiary structure. Then structure 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 relatively Initial event of the reaction is thought to be a relatively

uniform collapse of the protein molecule driven by uniform 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 nucleus Early formation of a diffuse protein folding nucleus

catalyzes further folding. The nucleus primarily consists of catalyzes further folding. The nucleus primarily consists of a few adjacent residues which have some correct 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 top Progress from the top to the bottom of the to the bottom of the funnel is funnel is accompanied by an accompanied by an increase in the increase in the native-like structure native-like structure as folding proceeds.as folding proceeds.

A new view of protein folding suggested that there is A new view of protein folding suggested that there is no single route, but a large ensemble of structures no single route, but a large ensemble of structures follow a many dimensional funnel to its native follow a many dimensional funnel to its native structure. structure.

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Protein Disulfide Isomerase (PDI)Protein Disulfide Isomerase (PDI)

The formation of correct The formation of correct disulfide pairings in nascent disulfide pairings in nascent proteins is catalyzed by PDI.proteins is catalyzed by PDI.

PDI preferentially binds with PDI preferentially binds with peptides that contain Cys peptides that contain Cys residues. residues.

By shuffling disulfide bonds, By shuffling disulfide bonds, PDI enables proteins to PDI enables proteins to quickly find the quickly find the thermodynamically most thermodynamically most stable pairing that are stable pairing that are accessible.accessible.

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Peptidyl Prolyl Isomerase (PPI)Peptidyl Prolyl Isomerase (PPI)

Prolyl isomerization is the Prolyl isomerization is the rate-limiting in the folding of rate-limiting in the folding of many proteins in vitro.many proteins in vitro.

PPI accelerates cis-trans PPI accelerates cis-trans isomerization more than 300 isomerization more than 300 fold by twisting the peptide fold by twisting the peptide bond so that the C,O, and N bond 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 fold Nascent polypeptides come off the ribosome and fold spontaneously; molecular chaperones are involved in spontaneously; molecular chaperones are involved in their folding in vivo, and are related to heat shock their 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))

hsp40 hsp40 hsp60 (e.g. GroEL in E. coli) hsp60 (e.g. GroEL in E. coli) hsp70 (DnaK in E. coli) hsp70 (DnaK in E. coli) hsp90 hsp90 hsp100 hsp100

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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 environmental dimensional shape when exposed to environmental extremes such as a extremes such as a low or high pHlow or high pH, or a , or a highly variable highly variable temperaturetemperature..

Changes in the pH of the proteins environment may alter Changes in the pH of the proteins environment may alter the charges on the amino acid side chains, altering the the charges on the amino acid side chains, altering the secondary and tertiary structure of the protein as a secondary 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 such Certain proteins, mainly digestive enzymes such as as trypsintrypsin, are capable of with-standing a , are capable of with-standing a pH as low as pH 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 molecular 3-Dimensional structure useful in molecular drug design.drug design.

Genome projects are providing sequences for Genome projects are providing sequences for many proteins whose structure will need to be many proteins whose structure will need to be determined.determined.

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Protein misfoldingProtein misfolding

If the protein misfolds, its properties can be If the protein misfolds, its properties can be markedly changed, as the way protein folds markedly changed, as the way protein folds determines which active groups are exposed for determines which active groups are exposed for interaction.interaction.

One example of this is in Transmissible One example of this is in Transmissible Spongiform Encephalopathies, such as Spongiform Encephalopathies, such as BSEBSE, and , and ScrapieScrapie. .

In these, the In these, the prion proteinprion protein, which is involved in the , which is involved in the brain'sbrain's copper metabolismcopper metabolism, misfolds, and starts , misfolds, and starts forming plaques, which destroy brain tissue. forming plaques, which destroy brain 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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QUERIES…

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