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Protein Folding
Proteostasis: The many fates of a protein
Maintenance#of#protein#ac6vity#is#
coordinated#by#many#pathways,#
involving:#
• Folding#• Unfolding#• Misfolding#
• Refolding#• Aggrega6on#• Degrada6on#
Protein Stability & Folding
In(vitro(folding(experiments,(or(more(commonly(unfolding(experiments(can(provide(insight(into(the(overall(stability(of(a(protein’s(3D>structure(
How&to&assay&protein&folding/unfolding?&#
Proteins#are#unfolded,#or#‘denatured’#by:#
• Temperature#(both#heat#and#cold)#
• pH#extremes#
• Organic#Solvents#• Chaotropic#Agents#
• e.g.,#urea#and#guandinium#hydrochloride#(GdnHCl)#
• These#disrupt#H?bonds,#and#prevent#hydrophobic#effect#
Typical#Experiment:#Monitory#Cataly6c#Ac6vity#and/or#Structure#vs.#Denaturing#Condi6on#
Urea& GdnHCl&
Protein Stability & Folding Example, Thermal Denaturation Monitor(Protein(Folding(vs.(Temperature(• For(example,(using(CD(Spectrometry(or(Tryptophan(Fluorescence(• Some(Proteins(are(very(stable(–(can(withstand(100°(C((e.g.(Archaea(Protein)(
Unfolding&Transi=on&Point&
Tm&=&“Mel=ng&Temp”&
Unfolding/Folding#is#Coopera,ve#
Protein Stability & Folding Example, Thermal Denaturation Denatura8on(with(Heat(will(oVen(lead(to(precipita8on(–(irreversible((
• Therefore,(assays(using(chaotropic(agents(or(detergents(may(provide(more(useful(biochemical(tools((
Protein Stability & Folding Example, Chaotropic Denaturation Monitor(Protein(Folding(vs.([GdnHCl](• For(example,(using(CD(Spectrometry,(Fluorescence(or(NMR(Spectroscopy(• Denatura8on/Structure(can(be(monitored(experimentally((
$ Insight(into(folding(mechanism(
Protein Stability & Folding In Vitro Refolding Experiments Some&proteins&can&be&‘reEfolded’&following&in&vitro&denatura=on&&&
(Typically(smaller(proteins(with(rela8vely(simple(na8ve(conforma8on)(
Example,&Ribonuclease:&• Small#protein#(13.7#kDa);#8#cysteines#(4#disulfides)#
• Denature#with#Urea#and#β?mercaptoethanol#
• Remove#Urea#&#BME#via#dialysis,#or#dilu6on#
• Protein#spontaneously#refolds#&#S?S#bonds#reform##
&
Major&Discovery!&
Sequence#alone#determines#the#na6ve#conforma6on#
• Nobel#for#Chemistry#in#1972#for#Chris#Anfinsen#
#
Protein Folding Kinetics: Levinthal’s Paradox
Protein#Folding#is#Very(Fast(&#is#Not(Random(–#Consider:###
For#a#100aa#protein,#if#we#assume#protein#folding#is#random#
• Each#residue#can#adopt#~10#conforma6ons#
• This#gives#10100#possible#conforma6ons#
• Now,#if#each#conforma6on#is#sampled#for#10?13#second#(6me#of#a#bond#vibra6on)#
• It#would#take#1077#years#to#sample#all#possible#conforma6ons.##
o i.e.(1.5#billion,#billion,#billion,#billion,#billion,#billion,#billion,#billion#years!)###
Obviously,#protein#folding#is#not#random!##Recall,(E.coli(ribosome(can(produces(a(folded(100aa(protein(in(<(5sec!(#
How do proteins fold so fast?
The#search#for#na,ve#conforma,on#is#not#random##
Molten globule states
Folding&is&Hierarchal&
• local#secondary#structure#is#first#
####(local#h?bond#&#ionic#interac6ons#guide)#
• followed#by#interac6on#of#neighboring#
#####secondary#structure#elements#
• Con6nued#un6l#complete#
• May#involve#mul6ple#folding#pathways##
• Larger#protein#sequences#may#ini6ate#
######folding#at#N?terminus#while#being#
######synthesized#
#
Protein Folding Prediction
Protein Folding: Thermodynamic Free-Energy Landscape
“Free&Energy&Funnel”&&
Illustrates(the(thermodynamic(pathway(s)(of(protein(folding((• Start#at#high#free#energy#state(s)#
with#larger#amount#of#entropy#
• Movement#down#the#free#energy#
landscape#is#guided#by#favorable#
thermodynamics#at#cost#of#entropy#
• May#cross#several#folding#
intermediates,#un6l#a#free?energy#
minimum#or#minima#is#found##
Protein Folding: Thermodynamic Free-Energy Landscape
Mul6ple#‘random’#
folding#pathways##
• No#intermediates#
• Stable#na6ve?
structure#
Mul6ple#‘non?random’#
folding#pathways##
• Many#intermediates#
• Stable#na6ve?
structure#
Only#a#few#folding#
pathways##
• No#intermediates#
• Stable#na6ve?
structure#
Mul6ple#folding#
pathways##
• 1#stable#intermediate#
• Stable#na6ve?
structure#
Most#Common#
Protein Folding: Computational Simulation
Methods&for&Sequence&Based&
Protein&Structure&Predic=on&#
Ab&ini=o&Methods&
Based#on#Thermodynamic#Principles#
Only#
#
SemiEempirical&Methods&
Based#on#Thermodynamics#and#
Previous#Experimental#Results##
(i.e.(sta8s8cal(analysis(of(the(PDB,(co>varia8on(analysis,(etc.)(
#
Protein Folding: Computational Simulation
Cri=cal&Assessment&of&protein&Structure&
Predic=on&(CASP)&Compe==on&
&
BiEannual&worldEwide&compe==on&of&>100&
research&groups&&
• Provided&Amino&Acid&Sequence&
• Experimental&3D&Structure&Kept&Secret&
Un=l&Compe==on&is&Over&&
Several&Groups&are&Now&Very&Successful&for&Proteins&~100aa&&
(Successful(=(predic8on(is(within(a(few(Å’s(of(the(experimental(structure)(
Image#credit:#wiki#
Protein Folding: Computational Simulation - Rosetta
RoseOa@home&
Baker&Lab&–&University&of&Washington&&
“Borrow”&computers&
that&are¬&being&used!&
How(to(overcome(the(massive(computa8onal(requirements(of(complex(proteins?(#
Protein Folding: Computational Simulation - Rosetta
FoldEit&
Baker&Lab&–&University&of&Washington&&
Make&it&a&Game!&
How(to(overcome(the(massive(computa8onal(requirements(of(complex(proteins?(#
hSp://fold.it#
Protein Folding In Cells
Protein Folding
Many&proteins&fold&spontaneously&in&the&cell&
&
But,&how&to&fold&more&complex&proteins,&or&prevent&misEfolding?&
&
Use&other&proteins&to&help&–&i.e.(Chaperones&
Protein Folding: Chaperones
#
• Bind#exposed#hydrophobics#
• Prevent#aggrega6on#
• They#do#not#ac6vely#
promote#folding#
Hsp70Eclass&of&Chaperones((e.g.(Hsp(70/40(system)(
Protein Folding: Chaperonins
Chaperonins:&&ac=vely&facilitate&Folding,&or&(re)Folding&
(e.g.(GroEL/GroES(System)(
Mechanism&• Unfolded#protein#enter#a#
GroEL#electrosta6c#chamber#
• GroES#binds#GroEL,#closes#
chamber#
• 7#ATP’s#hydrolyzed#
• GroES#&#folded#protein#
released#
• Repeated#at#opposite#
chamber#
Protein Folding: Chaperonins
Mechanisms#are#determined#from#combina,on#of#func,onal#assays#and#structural#studies#
Chaperonins:&&ac=vely&facilitate&Folding,&or&(re)Folding&
(e.g.(GroEL/GroES(System)(
Protein Mis-Folding & Disease
Protein Folding & Disease
Protein#misfolding#is#common#in#living#cells##
In#healthy#cells,#misfolded#proteins#may#be#refolded#or#disposed#
of#by#degrada6on##
In#unhealthy#cells,#or#individuals#with#gene6c#disease#–#misfolded#
proteins#may#accumulate#and#lead#to#disease##
A#few#examples:#• Cataracts#and#blindness#• Alzheimer’s#disease#
• Diabetes#• Parkinson’s#disease#
Protein Misfolding & Disease
credit:#www.reviewofoptometry.com#
Cataracts:#Protein#Aggrega6on#Disease#Gene6c#Muta6on/#
Age?Related#Modifica6on#
“chaperone”#
Protein Misfolding & Disease
Amyloidosis:#Alzheimer’s,#Parkinson’s,#Type#II#Diabetes…#
Amyloid#Fibril#Forma,on#
• Secreted#protein#is#misfolded/par6ally#folded#
• Associate#into#β?sheet#fibrils#
• These#assemble#into#insoluble#aggregates#
credit:#wiki#
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