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&not&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#