Protein Folding.pdf

PRTEIN FOLDING

1. Introduction
Newlysynthesizedpolypeptidefoldsintoitscharacteristicand
functional3-Dstructureviaaphysicalprocessknownasprotein
folding.
Interactionsamongaaleadtoformationofafolded3-Dstructure
knownasnativeproteinwhichisstable.
Proteinsmustmaintainconformationalflexibilitytofunction
Continualmaintenanceofactivesetofcellularproteinsrequiredunder
givensetofconditionsiscalledproteostasis

2. Proteostasis pathways
Fig.4.25. Lehninger Principles of Biochemistry, 6
th
Ed.

3. Denaturation at protein levels
Atprimarystructure:Sequenceofaaheldtogetherbycovalent
peptidebonds,isnotdisruptedbydenaturation.
Atsecondarystructure:Proteinsloseallregularrepeatingpatterns
suchasα-helicesandβ-pleatedsheetsandadoptarandomcoil
shape.

4. Cont--
Attertiarystructure:Disruptionofcovalentinteractionsbetweenaaside
chains(suchasdisulfide-bridgesbetcysteinegroups),non-covalent
interactionsbetweenpolaraaside-chains,van-derwaalsinteractions
betweennon-polaraasidechains.
Atquaternarystructure:Proteinsub-unitsaredissociatedand/orspatial
arrangementofproteinsubunitsisdisrupted.

5. Loss of protein structure results in loss of function
Native Protein denaturate in presence of reducing agent, alter
pH, temp, ionic strength, and solubility.
Various denaturants are:
1.Heatdisruptshydrogenbondsandhydrophobicinteractionsbet
non-polarresidues.Ex.Albuminineggdenatureandcoagulate
duringcooking.
2.Strongacidsandbasesdisruptssaltbridgesformedinaprotein
structure.Ex.Indigestivesystem,acidicgastricjuicescauses
coagulationofmilkbyproteolyticenzymerenin.

6. Cont--
3.Reducingagentslikeguanidinehydrochloride(GdnHCl)orβ-
mercaptoethanolreducesdisulphidebondstosulfhydrylgroupand
breaksintraandinterchaindisulphidebonds
4.Organicsolvents,ureaanddetergents(SDS)disruptshydrophobic
interactionsthatstabilizecoreofglobularproteins
•Ureadisruptsstabilizinghydrophobicinteractions,thusfreeingentire
polypeptidefromitsfoldedconformation.
5.ExtremesofpHalternetchargeonprotein,causingelectrostatic
repulsionanddisruptionofhydrogenbonding

7. Amino acid sequence determines tertiary structure
3°structureofglobularproteindeterminedbyitsaasequence.Proof
ofthiscamefromexperimentsshowedthatdenaturationofsome
proteinsisreversible
Certainglobularproteinsdenaturedbydenaturingreagentswill
regaintheirstablenativestructureandbiologicalactivity.This
processiscalledrenaturation.
DenaturationandrenaturationofribonucleaseA,demonstratedby
ChristianBAnfinsengotNobelprizeinchemistry1972

8. Cont--
Heprovidedfirstevidencethataminoacidsequenceofapolypeptide
chaincontainsallinformationrequiredtofoldchainintoitsnative,3-D
structure.
3-Dstructureandfunctionofproteinsdestroyedbydenaturation,which
demonstraterelationshipbetweenstructureandfunction.
Somedenaturedproteinscanrenaturespontaneouslytoform
biologicallyactiveprotein.

9. Lehninger Principles of Biochemistry by David L Nelson
Renaturation of unfolded, denatured Ribonuclease

10. Protein folding in sequential manner
(Image by MIT Open Course Ware, adapted from image by Professor Jonathan
King)
1.Newlysynthesizedpolypeptide
chainemergesfromribosome,
shortsegmentsfoldinto
secondarystructuralunits

11. Cont--
2.Foldingproceedsviaaninitialclusteringamongsidechainsof
hydrophobicresiduewhichprefertobealooffromanaqueous
environment:
Clusteringduetonon-specificinteractionamonghydrophobicresidues
leadtoformationofacompactarrangement(moltenglobulestate)
Hydrophobicresiduesofproteinsgatherinsidecollapsedformswithin
core
Collapsedstatefavorsformationof2°structure&encourages3°
interactionamongresidues

12. Assisted Protein Folding
MostProteinsfoldspontaneouslytotheirnativebutsomeproteins
undergoassistedfolding
Foldingofsomeproteinsrequirechaperons,thesearespecialized
proteinswhichinteractwithpartiallyfoldedorimproperfolded
polypeptides,ensurecorrectfoldingpathways.Ex.Chaperonesi.e,Hsp
70familyandchaperonins
Finally,foldingpathwaysofsomeproteinsrequiretwoenzymes
(ProteindisulfideisomeraseandPeptideprolylcis-transisomerase)

13. Cont--
Chaperones:Hsp70familyofchaperonesmoreabundantincells
stressedbyelevatedtemperatures:
•Itbindstoregionsofunfoldedpolypeptidesrichinhydrophobic
residuesmaybreakupproteinaggregateortopreventformationof
newaggregate
•Itprotectbothproteinsbyheatdenaturation
•Itblocksfoldingofcertainproteinsthatremainsunfoldeduntilthey
translocatedacrossamembrane
•Itassistedfoldingandassembly

14. Cont--
2.Chaperonins:Chaperonesprovidemicroenvironmentsinwhich
foldingoccur:
•Theyareelaborateproteincomplexesrequiredforfoldingofsome
cellularproteinsthatdonotfoldspontaneously
•Hsp60actsaschaperoninsinfoldingprocess,togetherwithan
Hsp70chaperone
•CentralcavityofHsp60chaperoneprovidesashelteredenvironment
inwhichapolypeptidefolduntilallhydrophobicregionsareburiedin
itsinterior,thuspreventproteinaggregation.

15. Cont--
TwoenzymesinvolvedinProteinFoldingPathways
1)Proteindisulfideisomerase(PDI):
•Catalyzesinterchange,shufflingofdisulfidebondsuntilbonds
ofnativeconformationareformed
•Catalyzeseliminationoffoldingintermediateswithinappropriate
disulfidecross-links

16. Cont--
2)Peptideprolylcis-transisomerase(PPI):
•Catalyzesinterconversionofcisandtransisomersofproresidue
peptidebonds,whichcanbeslowstepinproteinfoldingthatcontains
somepropeptidebondsincisconformation

17. Chaperone-assisted protein folding
Fig.8.23: The Cell, 4
th
Ed

18. Protein Misfolding and Diseases
Incompletelyandincorrectlyfoldedproteinsleadstwoseriousproblems
tocells:
1. Loss of function due to absence of correctly folded protein:
•Cystic fibrosis
2. Aggregation of incorrectly folded proteins:
•Alzheimer’s disease (Amyloid beta)

19. Cystic Fibrosis (CFTR)
Cysticfibrosistransmembraneconductanceregulator(CFTR)isa
membraneproteinandencodedbyCFTRgene.
CFTRgenecodesforanABC(ATPbindingcassette)transporter-
classionchannelproteinthatconductschlorideionscrossepithelial
cellmembranes.
MutationsofCFTRgeneaffectchlorideionchannelfunctionleadsto
dysregulationofepithelialfluidtransportinlung,pancreasresultingin
cysticfibrosis.

20. Cont--
Causedbydeletionofa3nucleotideswhichresultsinalossofaa
(Phe)residueat508
th
position,causesimproperproteinfolding.
Improvedunderstandingofproteinfoldingmayleadtonewtherapies
forCFTR.

21. Alzheimer’s disease (Amyloid beta)
Refoldingormisfoldingofβ-amyloidproteinendogenoustohuman
braintissue.
Senileplaquesandneurofibrillarybundlescontainaggregatesof
proteinβ-amyloid.
A4.3KDapolypeptideproducedbyproteolyticcleavageofalarger
proteinknownasamyloidprecursorprotein(APP).

22. Cont--
Levelsofβ-amyloidbecomeelevated
Thisproteinundergoesaconformationaltransformationfroma
solubleα-helixtoβ-sheetandpronetoself-aggregation.

23. Summary
Inproteinfoldingsteps,regionsofsecondarystructuremayform,
followedbyfoldingintosupersecondarystructures.
Largeensemblesoffoldingintermediatesarerapidlybroughttoa
singlenativeconformation.
Formanyproteins,foldingisfacilitatedbyHsp70chaperonesandby
chaperonins.

24. Cont--
The3-Dstructureandthefunctionofproteinsdestroyedby
denaturation,whichdemonstratetherelationshipbetweenstructure
andfunction.
Somedenaturedproteinscanrenaturespontaneouslytoform
biologicallyactiveprotein.
Disulfidebondformationandcis-transisomerizationofPropeptide
bondsarecatalyzedbyspecificenzymes.

25. Reference Books
1)Harper’s Illustrated Biochemistry-30
th
Ed
2)Biochemistry 7
th
Ed by Jeremy M. Berg, John L. Tymoczko and
Lubert Stryer
3)Lehninger Principles of Biochemistry, 6
th
Ed.
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