2. Denaturation and renaturation of proteins
3. Steps of Protein Folding
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4. Assisted Protein Folding by Chaperons
5. Enzymes involved in Folding Pathways
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6. Protein Misfolding and related DiseasesIntroduction
Newly synthesized polypeptide folds into its characteristic and
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functional 3-D structure via a physical process known as proteinfolding.
Interactions among aa lead to formation of a folded 3-D structure
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known as native protein which is stable.
Proteins must maintain conformational flexibility to function
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Continual maintenance of active set of cellular proteins required undergiven set of conditions is called proteostasis
Proteostasis pathways
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Fig.4.25. Lehninger Principles of Biochemistry, 6th Ed.
Denaturation at protein levels
At primary structure: Sequence of aa held together by covalent
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peptide bonds, is not disrupted by denaturation.
At secondary structure: Proteins lose all regular repeating patterns
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such as -helices and -pleated sheets and adopt a random coilshape.
Cont--
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At tertiary structure: Disruption of covalent interactions between aa side
chains (such as disulfide-bridges bet cysteine groups), non-covalent
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interactions between polar aa side-chains, van-der waals interactionsbetween non-polar aa side chains.
At quaternary structure: Protein sub-units are dissociated and/or spatial
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arrangement of protein subunits is disrupted.
Loss of protein structure results in loss of function
Native Protein denaturate in presence of reducing agent, alter
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pH, temp, ionic strength, and solubility.
Various denaturants are:
1. Heat disrupts hydrogen bonds and hydrophobic interactions bet
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non-polar residues. Ex. Albumin in egg denature and coagulate
during cooking.
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2. Strong acids and bases disrupts salt bridges formed in a proteinstructure. Ex. In digestive system, acidic gastric juices causes
coagulation of milk by proteolytic enzyme renin.
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Cont--
3. Reducing agents like guanidine hydrochloride (GdnHCl) or -
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mercaptoethanol reduces disulphide bonds to sulfhydryl group andbreaks intra and interchain disulphide bonds
4. Organic solvents, urea and detergents (SDS) disrupts hydrophobic
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interactions that stabilize core of globular proteins
? Urea disrupts stabilizing hydrophobic interactions, thus freeing entire
polypeptide from its folded conformation.
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5. Extremes of pH alter net charge on protein, causing electrostatic
repulsion and disruption of hydrogen bonding
Amino acid sequence determines tertiary structure
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3? structure of globular protein determined by its aa sequence. Proof
of this came from experiments showed that denaturation of some
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proteins is reversibleCertain globular proteins denatured by denaturing reagents wil
regain their stable native structure and biological activity. This
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process is called renaturation.
Denaturation and renaturation of ribonuclease A, demonstrated by
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Christian B Anfinsen got Nobel prize in chemistry 1972Cont--
He provided first evidence that amino acid sequence of a polypeptide
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chain contains all information required to fold chain into its native, 3-D
structure.
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3-D structure and function of proteins destroyed by denaturation, whichdemonstrate relationship between structure and function.
Some denatured proteins can renature spontaneously to form
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biologically active protein.
Renaturation of unfolded, denatured Ribonuclease
Lehninger Principles of Biochemistry by David L Nelson
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Protein folding in sequential manner
1. Newly synthesized polypeptide
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chain emerges from ribosome,short segments fold into
secondary structural units
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(Image by MIT Open Course Ware, adapted from image by Professor Jonathan
King)
Cont--
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2. Folding proceeds via an initial clustering among side chains of
hydrophobic residue which prefer to be aloof from an aqueous
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environment:Clustering due to non-specific interaction among hydrophobic residues
lead to formation of a compact arrangement (molten globule state)
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Hydrophobic residues of proteins gather inside collapsed forms within
core
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Collapsed state favors formation of 2? structure & encourages 3?interaction among residues
Assisted Protein Folding
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Most Proteins fold spontaneously to their native but some proteins
undergo assisted folding
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Folding of some proteins require chaperons, these are specializedproteins which interact with partially folded or improper folded
polypeptides, ensure correct folding pathways. Ex. Chaperones i.e, Hsp
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70 family and chaperonins
Finally, folding pathways of some proteins require two enzymes
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(Protein disulfide isomerase and Peptide prolyl cis-trans isomerase)Cont--
Chaperones: Hsp70 family of chaperones more abundant in cells
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stressed by elevated temperatures:? It binds to regions of unfolded polypeptides rich in hydrophobic
residues may break up protein aggregate or to prevent formation of
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new aggregate
? It protect both proteins by heat denaturation
? It blocks folding of certain proteins that remains unfolded until they
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translocated across a membrane
? It assisted folding and assembly
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Cont--2. Chaperonins: Chaperones provide microenvironments in which
folding occur:
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? They are elaborate protein complexes required for folding of some
cellular proteins that do not fold spontaneously
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? Hsp60 acts as chaperonins in folding process, together with anHsp70 chaperone
? Central cavity of Hsp60 chaperone provides a sheltered environment
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in which a polypeptide fold until all hydrophobic regions are buried in
its interior, thus prevent protein aggregation.
Cont--
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Two enzymes involved in Protein Folding Pathways
1) Protein disulfide isomerase (PDI):
? Catalyzes interchange, shuffling of disulfide bonds until bonds
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of native conformation are formed
? Catalyzes elimination of folding intermediates with inappropriate
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disulfide cross-linksCont--
2) Peptide prolyl cis-trans isomerase (PPI):
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? Catalyzes interconversion of cis and trans isomers of pro residue
peptide bonds, which can be slow step in protein folding that contains
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some pro peptide bonds in cis conformationChaperone-assisted protein folding
Fig.8.23: The Cel , 4th Ed
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Protein Misfolding and DiseasesIncompletely and incorrectly folded proteins leads two serious problems
to cells:
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1. Loss of function due to absence of correctly folded protein:
? Cystic fibrosis
2. Aggregation of incorrectly folded proteins:
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? Alzheimer's disease (Amyloid beta)Cystic Fibrosis (CFTR)
Cystic fibrosis transmembrane conductance regulator (CFTR) is a
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membrane protein and encoded by CFTR gene.CFTR gene codes for an ABC (ATP binding cassette) transporter-
class ion channel protein that conducts chloride ions cross epithelial
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cell membranes.
Mutations of CFTR gene affect chloride ion channel function leads to
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dysregulation of epithelial fluid transport in lung, pancreas resulting incystic fibrosis.
Cont--
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Caused by deletion of a 3 nucleotides which results in a loss of aa
(Phe) residue at 508th position, causes improper protein folding.
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Improved understanding of protein folding may lead to new therapiesfor CFTR.
Alzheimer's disease (Amyloid beta)
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Refolding or misfolding of -amyloid protein endogenous to humanbrain tissue.
Senile plaques and neurofibril ary bundles contain aggregates of
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protein -amyloid.
A 4.3 KDa polypeptide produced by proteolytic cleavage of a larger
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protein known as amyloid precursor protein (APP).Cont--
Levels of -amyloid become elevated
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This protein undergoes a conformational transformation from a
soluble -helix to -sheet and prone to self-aggregation.
Summary
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In protein folding steps, regions of secondary structure may form,
followed by folding into supersecondary structures.
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Large ensembles of folding intermediates are rapidly brought to asingle native conformation.
For many proteins, folding is facilitated by Hsp70 chaperones and by
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chaperonins.
Cont--
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The 3-D structure and the function of proteins destroyed bydenaturation, which demonstrate the relationship between structure
and function.
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Some denatured proteins can renature spontaneously to form
biologically active protein.
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Disulfide bond formation and cis-trans isomerization of Pro peptidebonds are catalyzed by specific enzymes.
27
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Reference Books1) Harper's Il ustrated Biochemistry-30th Ed
2) Biochemistry 7th Ed by Jeremy M. Berg, John L. Tymoczko and
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Lubert Stryer3) Lehninger Principles of Biochemistry, 6th Ed.
Thank you
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