The use of SAXS to study biological relevant systems

UNIVERSIDADEDESÃOPAULO
INSTITUTODEFÍSICA
TheuseofSAXStostudybiological
relevantsystems
LeandroR.S.Barbosa
[email protected]
2016
Laboratório de Biofísica!
EquipamentosdeApoio
[email protected]
Calorímetro Diferencial de Varredura
DMPC
DPPC
DSPC
Viscosímetro de Ostwald
η = kt
Schott - ViscoClock
3.5
t
η relativa
3.0
2.5
2.0
1.5
1.0
Capilar de Ostwald
0
5
[email protected]
10
15
20
25
30
35
40
Temperature(ºC)
45
50
55
Ressonância Paramagnética Eletrônica
Espectro EPR
Mobilidade–Microambiente
[email protected]
B (G)
Espectrofotômetro UV-Visível
2.0
Determinação da concentração de
proteínas;
Absorbância
Estrutura Eletrônica
...
1.6
1.2
0.8
0.4
0.0
280
300
320
340
360
λ (nm)
380
400
420
440
Fluorescência Estática e Resolvida no Tempo
-1
3
-1
λ (x10 cm )
26.7 25.0 23.5 22.2 21.0 20.0 19.1 18.2 17.4 16.7
1000
Intensidade Normalizada
Contagens
1.0
100
10
0.0
5.0
10.0
15.0
t (ns)
20.0
25.0
30.0
16.0 15.4
Água
Metanol
Acetonitrila
Diclorometano
Clorofórmio
Ciclohexano
0.8
0.6
0.4
0.2
0.0
375
400
425
450
475
500
λ (nm)
525
550
575
600
625
650
Espalhamento de Raios-X a Baixos Ângulos
2.5
1E -3
1.5
p(r)(a .u.)
In te n s ity (a .u .)
T c O Y E
2.0
1.0
1E -4
0.5
0.0
1E -5
0
20
40
60
80
r(A )
1E -6
0.0
0.1
0.2
0.3
0.4
-1
q(A )
0.5
0.6
[email protected]
Comparisonamongstructuraltechniques.
Small-AngleX-RaySca/eringAppliedtoProteinsinSolu9on.
Barbosa,LRS,Spinozzi,F.Mariani,P.andItri,R.49–72,“ProteinsinSoluYonandat
Interfaces:MethodsandApplicaYonsinBiotechnologyandMaterialsScience”,2013,Wiley
SAXS Setup
Detector
Monocromator
2θ
RXSource
PolicromaYc
X-ray
CollimaYon
system
SampleHolder
Vacuumchamber
Beamstopper
Low-pressureSystem
q
2θ
Incoming RX: kin
Outcame X-ray: kout1
Sample Holder
Reciprocal
space
! 4π
q =
sin θ
λ
“Typical” SAXS Experiment
X-Ray
FormFactor
InterferencefuncYon
relatedtotheg(r)
SAXS Theory
∞
⎧⎪ 2
⎫⎪
2
2 sen(qr)
I(q)= n p ⎨〈 F (q)〉 + 4π n p 〈 F (q)〉 ∫ (g(r)−1)r
dr ⎬
qr
⎪⎩
⎪⎭
0
2 ∞
⎧
⎫⎪
〈 F (q)〉
⎪
2
2 sen(qr)
= n p 〈 F (q)〉 ⎨1+ 4π n p
(g(r)−1)r
dr ⎬
2
∫
qr
〈 F (q)〉 0
⎪⎩
⎪⎭
!"
!" "
= n p ( Δρ )V 2 P(q)S(q)
i qi r
Δρ = ρ Particle − ρ Solvent
F (q) =
∫ (ρ
Particle
( r ) − ρ Solvent ) e
dr
VParticle
For non interacting systems, S(q) = 1 describing the electron
density is to solve the problem!
For
non interacting systems, S(q) = 1 describing the electron
Example:HomogeneousSpherewithradiusRandconstant
density
is to solve the problem!
electronicdensity
! ⎧⎪ ρ
ρ r =⎨ S
⎪⎩ ρ 0
()
r≤R
r>R
⎧
I (q) ∝ ⎨ ∫
⎪V
⎩
( () )
2
! ! !⎫
!
ρ r − ρ 0 eiq⋅r d r ⎬
⎪
⎭
⎧
⎫
⎪
⎪
2 sen(qr)
= ⎨4π ( ρ E − ρ 0 ) ∫ r
dr ⎬
qr
⎪
⎪
0
⎩
⎭
R
2
⎧
⎧ sen(qR) − qRcos(qR) ⎫ ⎫
⎪
⎪
P(q) = ⎨3V ( ρ E − ρ 0 ) ⎨
⎬⎬
3
(qR)
⎪
⎩
⎭⎪
⎩
⎭
2
Asfarasyoudefinetheelectrondensityitispossibletocalculatethe
theoreYcalscacering...But,doesthiswork?
HomogeneousSphere(Latexspheres)
⎧ sen(qR) − qRcos(qR) ⎫
I (q) ∝ ⎨
⎬
3
(qR)
⎩
⎭
2
Figureextractedfrom:hcp://iramis.cea.fr/scm/lions/techniques/saxs/
Example:HomogeneousEllipsoidwithsami-axisR,RandvRand
I
constantelectronicdensity
⎧
x 2 + y 2 )ν 2 + z 2
(
⎪ρ
if
≤1
2 2
ρ ( x, y, z ) = ⎨ Particle
ν R
⎪ρ
otherwise
⎩ 0
⎧
I (q) ∝ ⎨ ∫
⎪
⎩V
π
PEllipsoid (q, R,υ ) =
2
∫
0
(
⎧
⎧
⎪
⎪ sin ( qR1 ) − qR1 cos(qR1 ) ⎫
⎪⎫
⎪
⎨3V ( ρ E − ρ 0 ) ⎨
⎬ ⎬ cos α d α
3
(qR1 )
⎪
⎪
⎪
⎩
⎭⎪
⎩
⎭
2
R1 = R sin α + ν cos α
2
2
( () )
2
! ! !⎫
!
ρ r − ρ 0 eiq⋅r d r ⎬
⎪
⎭
2
)
1
2
HomogeneousEllipsoid:Lysozyme
Lysosyme
Lysozyme 7 mg/mL
100
Ellipsoid of revolution + background
R = 15.48 Å
2=2.4 (χ=1.55)
χ
ε = 1.61 (prolate)
-1
I(q) [cm ]
10-1
10-2
10-3
0.0
0.1
0.2
q [Å-1]
0.3
0.4
19
Figureextractedfrom:FormandInterferencefactors:ModelingandinteracYons,J.S.Pedersen.
RadiusofgyraYon:InformaYonaboutthearrangementof
maceraroundthecenterofmass.
TheGuinier’sLaw:
describesthescacering
intensityintheq->0
limit…
n
1
2
R = ∑ ri
n i=1
2
g
Guinier’sLaw
1911-2000
I (q → 0)  I (0)e
−
q 2 Rg2
3
Åln[I (q)] = ln[I (0)] −
q 2 Rg2
3
!!if qRg ≤ 1.3!!
Rg = 26.0 ± 0.5 Å
Isthisimportantfor
whatkindofstudy?
Guinier’sLaw–Effectofureaonproteins:HIPcase
CDFluorescence
-Gnd-HClshowedtwodis9ncttransi9onswithCD(at
1.0and3.0M)andalsowithfluorescence(at0.9and
2.6M),whereasthecurvesusingureashowedonly
one(2.6M)welldefinedtransi9on.
Dores-Silvaetal.Arch.ofBiochemandBioph.,520,88-98,2012.
Andtheradiusofgyra:on?
Theradiusofgyra:onfollowsthesametrendofCDand
Fluorecence.ButisSAXSaspectroscopictechnique?
Dores-Silvaetal.Arch.ofBiochemandBioph.,520,88-98,2012.
Sistemasderelevânciabiológica
Proteínas;
Lipídeos;
Ácidos
Nucleicos;
Sacarídeos
[email protected]
Proteínas
•  Macromoléculasquedesempenhamosmais
diferentespapeisnascélulas/organismos:
•  Enzimas,hormônios(insulina),anYcorpos,
pepwdeos,...
•  Sãoformadasporumasequênciade
aminoácidos...
[email protected]
Aminoácidos
Todasasproteínassãoformadasporumasequêciade
aminoácidos.
Existem20Yposdeaminoácidose“Todos”apresentam
estaestrutura
[email protected]
[email protected]
Ecomoosaminoácidosseorganizam?
[email protected]
Ecomoosaminoácidosseorganizam?
EstruturaPrimária
EstruturaSecundária
EstruturaTerceária
EstruturaQuaternária
[email protected]
TécnicasFísicas:
Fluorescência,SAXS,
Espalhamentode
Luz...
Comosedáoprocessodeorganizaçãodos
aminoácidos?
Energia,
Entropia
RelaçãoEstrutura-Função
E0,S0
MínimodeEnergia
EdeEntropia
Masasvezesissonãoacontece…
[email protected]
A2aleidaTermodinâmicanosdizque…
“A quanFdade de entropia de um sistema
qualquer, isolado termodinamicamente,
tendeaaumentarparaumvalormáximoaté
que o equilíbrio termodinâmico seja
alcançado”
“AEntropiadoUniverso
devesempreaumentar” Energia,
Entropia
Masateoriadofunilde
envelamentoestariaviolando
asegundaleidatermodinâmica?
MínimodeEnergia
E0,S0
EdeEntropia
Masasvezesissonão
acontece…
AProteínapodeassumirestruturasestáveisdiferenteda
naYva?
Estados
Intermediários
SvenFrokjaer&DanielE.Otzen.
Proteindrugstability:aformulaYonchallenge
NatureReviewsDrugDiscovery4,298-306(2005)
EfeitodaTemperaturana
EstruturadeProteínas
AProteínapodeassumirestruturasestáveisdiferenteda
naYva
Masqualseriaaestruturamaisestável?
ThomasR.JahnandSheenaE.Radford
TheYinandYangofproteinfolding
FEBSJournal272(2005)5962–5970
SalahuddinP.ProteinFolding,Misfolding,AggregaYonAndAmyloid
FormaYon:MechanismsofA��OligomerMediatedToxiciYes.
JournalofBiochemistryandMolecularBiologyResearch2015;1(2):
36-45
AProteínapodeassumirestruturasestáveisdiferenteda
naYva
MaldeAlzheimer,
Parkinson
DoençadaVaca-Louca
DoençadeCreutzfeldt–Jakob
Estudodasformaçõesde
FibrasAmiloides
AsFibrasamiloidessão
ricasemestruturasdo
9poβ
[email protected]
UsingSAXStoelucidadetheamyloidfibrilformaYon
Parkinsonaffectsmorethen4mipeopleItheworld;
LostofdopaminergicneuronsintheSubstanFaNigra(regionofparsreFculata);
Some amyloidosis
1
2
Disease
Alzheimer'sdisease
AorYcmedialamyloid
Proteinfeatured
Betaamyloid
Medin
Abbrev.
Aβ
AMed
3
Atherosclerosis
ApolipoproteinAI
AApoA1
4
Cardiacarrhythmias,Isolatedatrialamyloidosis
AtrialnatriureYcfactor
AANF
5
Cerebralamyloidangiopathy
Betaamyloid
Aβ
6
Cerebralamyloidangiopathy(Icelandictype)
CystaYn
ACys
7
8
Diabetesmellitustype2
Dialysisrelatedamyloidosis
IAPP(Amylin)
Beta-2microglobulin
AIAPP
Aβ2M
9
Familialamyloidpolyneuropathy
TransthyreYn
ATTR
10
11
FatalFamilialInsomnia
Finnishamyloidosis
PrPSc
Gelsolin
APrP
AGel
12
Hereditarynon-neuropathicsystemicamyloidosis
Lysozyme
ALys
13
14
HunYngton'sDisease
La•cecornealdystrophy
HunYngYn
Keratoepithelin
none
AKer
15
Medullarycarcinomaofthethyroid
Calcitonin
ACal
16
17
18
Parkinson'sdisease
ProlacYnomas
RheumatoidarthriYs
Alpha-synuclein
ProlacYn
SerumamyloidA
none
APro
AA
19
SporadicInclusionBodyMyosiYs
S-IBM
none
20
systemicALamyloidosis
ImmunoglobulinlightchainAL
AL
21
Transmissiblespongiformencephalopathy“MadCow”
PrPSc
APrP
C.Dobson,NatureReviewsDrugDiscovery2,154-160(2003)
General model for Fibril formation
Besidesα-syn,otherproteinsarefound
intheamyloidfibrils,likeGAPDH…
h/p://lashuel-lab.epfl.ch/page-50760-en.html
Glyceraldehyde3-phosphate
dehydrogenase
Amyloidspreading
NormalNeuron
Ø  α-synisdegradedbyUPS
(ubiquiYn–proteasomesystem)(or
autophagy);
Ø  Small fracYon is released (exocy
tosis);
DiseasedNeuron
Ø 
Ø 
Ø 
Ø 
α-synisNOTdegradedbyUPS(norautophagy);
Largeamountsarereleased(exocytosis);
InternalizaYonbyneighboringcells;
The“seeds”cansurviveiflysosomalfuncYonis
impairedduetoageingorgeneYcmutaYons.
Leeetal.NatureReviewsNeurology10,92–98(2014)
General model for Fibril formation
Theoryofproteinfolding
tic because they tend
r (Fig. 1). This is due
ophobic amino-acid
ckbone to the solvent
Like intramolecular
c forces and primarily
ly, fibrillar aggregates
REVIEW INSIGHT
Thefunneltheory
Unfolded
Chaperones
Chaperones
Energy
g. 1). Chain collapse
native interactions
ds to be searched en
surface that must be
molecules must cross
nsequence, partially
s kinetically trapped
r proteins larger than
which have a strong
nto compact globular
isorganized globules
figurational entropy
-native interactions
ch for crucial native
eed, whereas in the
ay be rate-limiting1
obular intermediates
larger, topologically
by many long-range
ch proteins are often
GAPDH
Folding
intermediates
Partially
folded
states
Native
state
Oligomers
Amorphous
aggregates
Amyloid
fibrils
Intramolecular contacts
Intermolecular contacts
Figure 1 | Competing reactions of protein folding and aggregation. Scheme
of the funnel-shaped free-energy surface that proteins explore as they move
towards the native state (green) by forming intramolecular contacts (modified
from refs 19 and 95). The ruggedness of the free-energy landscape results in
the accumulation of kinetically trapped conformations that need to traverse
free-energy barriers to reach a favourable downhill path. In vivo, these
steps may be accelerated by chaperones39,41,42. When several molecules fold
Heparin
Amyloid formation of GAPDH and Heparin
THTfluorescence
λ=1617cm-1
SAXS data
Rg = 32(2) A
Rg = 50(1) A
Rg = 66(1) A
Guinier’slaw
I (q → 0) = I (0) e
− Rg2 q 2
3
sin ( qr )
r2
p (r ) =
I
q
4π q 2 dq
(
)
3 ∫
qr
( 2π ) V
PDDF
Torres-Bugeau,etal.2012,JBC,287,2398-2409
FIGURE 4. SAXS curves and data analysis of GAPDH in the presence and in
the absence of heparin. A, correspondence between theoretical and exper-
(Fig. 4C).
particle c
than Dma
SAXS res
bined to
indicated
Size Di
tion in th
at 37 °C a
Fig. 5A. P
dynamic
compatib
GAPDH
with a me
heparin (
average m
respective
was disca
nal could
Glycol
the nativ
bation w
(Fig. 5B).
tions coe
tetramer
Accord
GAPDH
for a non
of the hy
Thus, it i
arranged
(38, 52) t
where Rh
ments, an
respective
SAXS data
Time evolution of amyloid fibril formation
Systemissupposedtobecomposedof:
Ø NaYvetetramers;
Ø Dimers;
Ø Monomers
Ø Protofibrils(effecYvecylinders)
SAXS data
Nomonomerswereevidenced
Àvila,etal.2014,JBC,289,13838-50
Cylinder:2R=118(2)Å
L=215(9)Å
SAXS data and Molecular Dynamics Simulations
SAXSdatausedas“input”intheMDsimulaYons
Theprotofibrilsarecomposedof
“trimersofdimers”
Mechanism of fibril formation
Acknowledgements
Ø  Ins9tutodeQuímica/USP-SC
JulioCesarBorges
Ø  UNICAMP-IQ
CarlosH.I.Ramos;
Ø  Ins9tutoAdolfoLutz
AndréTempone
Ø  Ins9tutodeQuímica/USP-RP
PietroCiancaglini
Ø  F.deC.Farmacêu9cas/USP
CarlotaRangel
Ø  IFUSP
RosangelaItri
Ø  U.DeLisboa
NunoSantos
Ø  Ancona,Itália
PaoloMariani
FrancescoSpinozzi
Ø  IFUSP–Grupode
Biousica
MariaTeresaLamy