prot interaction

7/29/2019 Prot Interaction

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Chem/Biol 8360; Fall Semest

ass : ec n ques : pro emicroalorimetry, surface plasm

September 27, 2011

Instrumental MetProtein-Ligan

Lecturer: Bob Wohlhueter; bob

r 2011 (mini-semester I)

n- gan n erac on,n resonance, light scattering

ods for MeasuringInteractions

[email protected]


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Some useful references:

1. Biomolecular Ligand-Receptor BindinCharles R. Sanders, Dept. of Biochemistr

. .This is an excellent introduction to theory

2. Protein-Ligand Interactions: HydrodyB.Z. Chowdhry, eds.; Oxford University PrThis Practical Approach text, along withSpectroscopy cover all instrumental met

. ro e n- ro e n n erac ons: e o s Press (2004).This text covers mostly biological method

ha e-dis la not addressed in this lectu

4. Biocalorimetry, J.E. Ladbury and B.Z.

This text remains the authoritative sourcevery detailed, mathematically rigorous.

Studies: Theory, Practice, and Analysis, Vanderbilt University (available on-line at

.nd practice of binding measurement.

amics and Calorimetry, S.E. Harding andess (2001)ts companion volume on Structure andods in detail.

an pp ca ons , a an u, e .; umana

, like the yeast two-hybrid approach ande.

Chowdhr , eds.; Wiley (1998)

for theory and practice of microcalorimetry;


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General Introduction:1. Biological systems characteristicall

(H-bonds, hydrophobic bonds, electheir functioning.

a. sma mo ecu es n erac ng w mallosteric effectors, 2nd messenger

b. macromolecules interacting with e,pathways

2. Accordin l our abilit to understa

requires us to discern such interact

3. Not surprisingly, a large portion of tdirected at detecting non-covalent i

a. Qualitative (yes-or-no) methodsm croarrays, an exp o e n any

b. Quantitative methods for measurinradioisotopically) binding to protei

.mass, binding enthalpy) of the bindinstrumentation.

y rely on non-covalent interactionsrostatics, van der Waals forces) for

cromo ecu es, e.g. enzyme su s ra es,, promoters, membranes

ch other, e.g. enzyme complexes,, ,

d biolo ical structure and function

ions, often quantitatively.

he biochemists methodology is

nteractions.

sing bait-and-prey, implemented inorm o a n y c roma ograp yg small ligands (labeled, usuallys; typically equilibrium dialysis

,ing entities, and thus requiring


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Familiar examples of compl

dc

s

Udhat

T

togr

D.Voet and J.G.Voet, Biochemistry(

x, catalytic machinery:

nzyme complexes: pyruvateehydrogenase includes 26 subunitsomprising three catalytic activities;s n erme a es o no m x w uolvent.

iquitin system for proteingradation takes place in the

,llow tube, 190 x 450 , comprisingleast 3 catalytic functions.)

e ribosome, with 52 proteins and. ,al of 2.52 megaDaltons, is thend-master of ensemble function.

nd ed.), Wiley 2004)


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Caveat #1: the cell is not* It is a crowded, organized space, a 20

onsequences or v scos y, us on co

* Biochemists who like to work with purifishould alwa s bear this in ind.

B.Alberts et al.Molecular Biology of the Cell (

dilute, aqueous solution:protein solution.

ns an s, c em ca ac v y o 2

d proteins in dilute, aqueous solutions

th ed.), Garland (2002)


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Caveat #2: Beware of pro

Bait and Prey methods-

have been devised in which a bait p

of cellular proteins. Those that stick, t,

A recent protein-graph analysis [DePNAS 103: 311 2006 concludes that

by the yeast 2 hybridtechnique, therethe number of interactions made by aresidues on its surface.

Using a clever protocol [quantitativ

knockdown, QUICK], Selbach and Mas owe a , o pro e ns cap ureall but two were noise. To differentialabeled proteins in vivowith heavy- or

ein stickiness when using

,otein is exposed to a large collections

he prey, are construed having. . . . .

ds, Ashenberg and Schaknovich,in two ma or studies of east roteins

is a significant correlation betweenrotein and the fraction of hydrophobic

e immunoprecipitation combined with

nn [Nature Methods 3:981 (2006)]as prey y an o y- oun - ca en n,e signal from noise, the researcherslight- isotopes of N in normal and

.


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Physical Methods for Analysis

TechnologyPhysical QuantityMeasured

so erma ra oncalorimetry

enthalpy of reaction

-resonance

solid interface

-

scattering

photons

analytical sedimentation inultracentrifugation gravitational field

li ht s ectroscoabsorbance,fluorescence, Raman,circular dichroism

mass spectrometry mass of complex

f Macromolecular Interaction

Characteristics of Method

solution- hase bindin of macromoleculewith ligand; allows complete thermodynamiccharacterization

detects solution-phase ligand binding to-

immobilized molecule; kinetics ofassociation/dissociaton

computation of molecular size and shape inso u on; m e o macromo ecu arassociations

computation of molecular mass in solution;limited to macromolecular associations

dependent on ligand altering optical behavior

mass spectrometry occurs in vacuo;extrapolation to liquid phase?


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Technique 1: Calorimetryso er a ra on vs.

Note that in differential scanning cachamber is closed. One applies heat

rise in temperature as a function of hlinearity are due to transient changesas regions in which the protein undere.g. unfolding.

In isothermal titration calorimetry (Ireaction chamber is introduced conti

concentration of added ligand).

Accordin l DSC lends itself to roligand interaction studies.

eren a cann ng

lorimetry (DSC), the reactiono the chamber and measures the

at applied. Positive diversions fromin heat capacity, and are construedoes gross conformational change,

C), reagent extraneous to theuously. The heat generated (or

tein foldin studies ITC to rotein-


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Isothermal Titration Calorimetr

One rea

(ca. 100undergo

Secondstirred

ea sinstrume(set bet

Raw dat

(exother

the reacIntegratthese dto which

estimateH, Kd,

: basic experimental scheme

tant li and is contained in s rin e

l) at high concentration; its a 15-fold dilution upon injection.

eactant (protein) is preloaded in aell (1.5 ml).

n ca a e or w rawn ynt to maintain constant temperatureeen 2 and 80); the amount added is

.

is collected as cal/sec; integrated

mic) or taken up (endothermic) by

tion, as triggered by one injection.ed over time of whole experiment,ta describe a binding isotherm,a theoretical curve is fit to yield

s o t e t ermo ynamics parametersas well as reaction stoichiometry.


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ITC: example of raw and inte rated thermal data

Each spike represents asuccessive injection of ligandsolution, say 5 l.

As the binding reaction saturates,

area under the spike decreases.e sma , res ua sp es a ersaturation are construed as heatof dilution (of ligand solution in

,as correction.

as the integrated heat release

against molar ratio of reactants,calculated their from knownconcentrations.

Here, half-maximal heat occurs

at approximate y mo ar ratio = ,implying 1:1 stoichiometry.


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The directly measured quantity isH; K can be evaluated from theisotherm. If you perform theexper men a severa , a comp e ethermodynamic description of thebinding reaction can be calculated.


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ITC: experimental design con iderations


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ITC:Y.V. Griko

-

Protein Sci

-lactalbu-

denaturestable, an

and drive

At highershown) itthe stabilinegativefrom an e

.

etal ion binding to a proteinnd D.P. Remeta, Energetics of solvent and

- ,

nce 8:554 (1999)

min is a Ca2+ binding protein; the

states. At 5 its conformation isCa2+ binding is modestly exothermic

b ositive entro .

emperatures (only summary dataconformation is much more fluid, and

zation induced by Ca + involves a large,ntropy. That is, the reaction switchesthalpy-driven to an entropy-driven


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ITC: Ca2+-dependent peptide:A.Y. Gribenko, M.Guzman-Casado,M.M.Lopez and

thermodynamic properties of peptide binding

11:1367 (2002).

rotein interactionG.I. Makhatadze, Conformational and

o human S100P protein, Protein Science

- 2+,binding protein whoseexpression is elevated in

rostate cancer. It has bothhigh (Kd= 5 M) and low (Kd= 200 M) affinity calciumsites.

Mellitin is a 26-residuepeptide from bee venom with

-aqueous solution. The

characteristics of binding toon occupation of the low-affinity calcium site.

ITC C 2 d d id i i i ( )


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ITC: Ca2+-dependent peptide

capacity: Cp = [H0/ dT]P , and that aa conformational change in protein.

:protein interaction (cont)

hange in heat capacity usually implies


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Another example of ITC: T-c

K. M. Armstrong and B.M Baker, A comprehe

-

interation, Biophysical J. 93:597 (2007)

ll receptor:peptide binding

sive calorimetric investigation of an

MHC (type I) presents

antigenic peptides (ca. 10-mers) arising from theintracellular proteolysis ofviruses. The MHC:peptidecomp ex s recogn ze y aT-cell receptor specific tothe peptide. When the T-

infected cell, it kills the cell.

The A6 TCR reco nizesLLFGYPVYV generatedfrom the tax protein ofHTLV-1


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Binding of A6-T-Cell-- -

presenting HLA-A2

measured in this experiment (pH6.4)differed from those measured at

. ,suspect proton linkage with binding,i.e. that a shift in pKa occurs uponbindin .

They pursue this hypothesis bycarrying out the reaction in variousbuffers covering a wide range of

ionization enthalpies: protonsoun or re ease y pro e n are

released or bound by buffer.


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pH=6.4 Deon

impKa

I

o

pH=7.4toa

Tn

u

endence of binding enthalpythe bu er-t e i lies

lies that the binding altersof the protein.

deed, there is a correlation between

bserved enthalpy of the rxn (H0, one ordinate) the enthalpy of ionization

f the buffer used (Hib, on thebscissa).

he slopes of the lines give nH+, theumber of protons released by buffer;

i b pure H0 of protein:protein

ncomplicated by buffer interactions.

ere nH+ is -0.28 / molecule of protein,H0 = -1.15 kcal/mole


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pH=6.4

pH=7.4

Overall, this thorough studyallows conclusions o an

entropy-driven reaction,involving change in protein pKa

The dependence of H0obs ontemperature indicates a small

.

pprotonization.

Extendin such studies to

systematic changes in pH,temperature and bufferionization enthalpy, and applyingglobal fits to the data, supportsthe conclusion that the TCR

binds to the HLA:peptidecomp ex w a s g yexothermic reaction, driven by alarge, positive change in entropy

adaptive movement of the TCRsCDR loops.)


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ummary:

1. Isothermal titration calorimetrybinding reaction heat lends its

characterization of binding reac

2. It is capable of analyzing proteiion ligands, as well as heterolog

3. Limited by instrumental sensitivrequires fairly large amounts ofexperimental design.

4. It measures only macro thermo

reaction, and does not handle w

because it directly measureself to exquisite thermodynamic

ions.

-ligand binding, including metalous protein-protein binding

ity of heat measurement, itprotein, and careful attention to

ynamic properties of a

ell micro properties such as .

T h i 2


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Technique 2:Surface Plasmon Resonance

(as implemented in BIAcore

These cartoons and several that follow are tafrom websitehttp://www.biacore.com/lifesciences

SPR arises when the electricvector of light resonates with

)

electrons + holes, e.g. ingold), thereby losing energy at

incidence. The angle isdependent on the refractive

index of the medium n .

The effect ofn falls off isdistance, related to

wavelength of the impinginglight. In the BIAcoreinstrument, the depth of the

en

binding phase (the dextranphase in CM5 chips) is 120

nm.

Within this phase theincrement in mass when amolecule bindsis sufficient to

change n, and thus the dipangle.


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BIAcore couples SPR to a bioc

The BIAcore instrument exploits this phdextran) and connects it with a microfluiligands across the surface. A binding emass an increment in refractive indexresonance angle is recorded as the signagainst time.

emically friendly environment:

nomenon by providing the SPR-dics system which can move testent represents an increment in. This consequent shift in

l, a sensorgram of the shift

B h d fl d ff l


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BIAcore sensor chip and microfl

nm dewhichcom a

Fluid flcompu

It is usminiatcolum

in thepasse

uidics as affinity column:

p layer of carboxymethy-dextran,ffers a hydrophilic, easily derivatizedrtment.

ow over the sensor surface ister-controlled through a system of

, .eful to think of the sensor cell as arized, affinity chromatography: one bindin artner is immobilized

ell, and a ligand (the test analyte) isthrough it in a mobile phase.

Th li id h dli d l


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The microfluidic system in fact defines fourseparate areas (columns), each of whichcontain a different immobilized ligand. Mob

,combinations of them in tandem.

In addition to the standard CM5, a varietyof other surfaces are available: old onlmonolayer carboxymethylated surface; a shlayer of CM-dextran; dextran with a low degcarboxylation.

lipophilic chip (L-1) allows assembly of lilayers and embedded proteins to simulatemembrane receptor interactions.

The liquid-handling moduleallows one to schedule a

ligands to be passed throughthe sensor cell.

This includes the reagentsneeded to immobilize a

substance to the cell in thefirst place.

ayile

ortee of

id


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A Variety ofA n tyMatrices are

chips offers avariety of surfaces

immobilizationchemistry and ligandt es.

Surface Plasmon

Resonance,

P. Anton van der Merwe

available from BIAcore.

Immobilizing a substance to a carboxylated dextran surface:


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Immobilizing a substance to a ca

NHS =- y roxysucc n m e

PDEA =2- 2- ridin ldithio ethaneamine.HCl

EDC =N-ethyl-N-(dimethylaminopropyl)carbodiimi

rboxylated dextran surface:

e

Immobilization chemistry is impl mented on-line


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Immobilization chemistry is impl

3.

1.

.

mented on-line

1. Activation of carboxyls by

EDC/NHS; the signal bulkrefractive index shift.

2. Accumulation of ligand indextran phase. Not

necessarily covalenta ac men ; p s e era e yadjusted to help accumulateligand by ion-exchange. RUbetween *s measured actual

mass immobilized.

3. Ethanolamine is injected to

carboxyls not reacted withligand.

4. All such fluid movements,including binding experimentsare programmed by means of

.

BIACore: experimental str tegies


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B ore exper mental strSPR angle shifts are recorded as Resonexperimentally to amount of protein boun

of 1 pg protein/mm2

(factors for DNA or glvolume of dextran phase one can calculat

teg esnce Units (RU), which have been related

to the dextran phase: RU of 1 masscans are different). From the effective

e that RU of 1 conc of 10 mg / L.

BIAcore: experimental design considerations


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BIAcore: experimental de

1. The ligand to be immobilized shouorganic moleculer, etc.) or chemic

ayers .

2. Ligands presented in the mobile p

serum).

3. Because the SPR response is propone can enhance sensitivity by im

. ,

Da) to a peptide (e.g. MW = 1,5for an IgG:peptide event if you iantibod in mobile hase.

4. Be aware that immobilization of

linkage of its primary amino grousome cases this effect has beenappear as heterologous binding ki

sign considerations

ld be pure (peptide, protein, DNA, smalllly defined (e.g. construction of lipid

ase may be heterologous (e.g. diluted

ortional to mass of the binding ligand,obilizing the lessmassive binding

= ,

0 Da) you get 100x more sensitivitymobilize the peptide and present the

protein, for example, via covalent

s to dextran-carboxyls may result in.hown to introduce artifacts thatetics.

BIAcore: experimental design considerations (cont)


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BIAcore experimental design

5. Generally you want to strip off bo(regeneration) in order to use a

a. quantitatively strips off bound

b. leaving immobilized ligand intdilute formic acid, or chaotrop

6. BIAcore lends itself to a lot of proELISA: e.g. immobilized streptavisecondary Ab binding and other t

considerations (con t)

nd material from the sensor cellsurface for many rounds of binding.

mobile-phase ligand, while

ct. (Try short bursts of 10 mM HCl,ic salts.)

ocols immunologists have applied indin; competition/inhibition;ndem binding experiments.

The mass transport proble :


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The mass transport problecore c p es gn presents a poten

one would analyze binding curvesreaction:

A + B A:B

-concentration of both are experimthe dextran phase is not instantankinetics sometimes a l :

Amobile Adextran + B A:B

If A is bulky and/or [B] is high, the firsand if you analyze the data applyi

constants you get will certainly b

:a mass transport pro em. ea y,

according to a simple bimolecular

ental givens.) But diffusion of A intoeous, so that in reality biphasic

step may become rate-limiting. If so,g the simple model, the rate-

wrong!

The mass transport proble : Remedies


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The mass transport proble

REMEDIES: the robust approach is to

order with respect both to [A] and [B].relatively easy: simply vary the concensystematically varying the effective coeasy. But there are work-arounds:

. Amobile Adextran + B A:B

slower and thus more likely to be r

2. Use BIAcores Pioneer Chip F1,phase, and thus is less likely to be

3. If possible, try reversing the rolesrate-constants in both configuratio

against orientation heterology.)

4. Try fitting more a complex kineticIs it a signifantly better fit to the da

allow such models.)

: Remedies

rove the reaction velocities are 1st-

With mobile-phase ligand A, this isration of A in mobile phase. Butcentration of immobilized B is not so

ate limiting.

hich has a much less dense dextranbarrier to As penetration.

f A and B. Do you get the sames? (This is also a strong argument

odel which contains a diffusion term.a? (BIAcores analysis software, and

. . .

The mass transport proble : sometimes its useful


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The mass transport proble

There are situations in which you wowrt [A] and zero-order wrt [B].

Amobile Adextran + Bimmobilized A:B

For example, if you are interested incomponen n serum, an on car

binding. In that case, immobilize a herate to be limiting in As diffusion into

: sometimes its useful

ld like the reaction to be 1st-order

stimating the concentration ofa ou measur ng e ne cs o

avy load of B to force the overallthe dextran phase.


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Evaluatin BIAcore Data:

General Considerations:

. core ec no ogy n eren y yequilibrium concentrations). Forconcentration of analyte [A], one

2. The reaction curves encompassof the binding reaction, not just igenerally wants to use rate equaanalyze the data.

3. Typically a kinetic run is done ovconcen ra on run y e s a u sekinetic parameters are over-det

globally, i.e. all together enhan

4. Consistency of fit parameters ovargument that the rate-law used i

e s ne c a a as we asany single run at a singlecan measure a k

on

, koff

, KD

nearly the complete time coursenitial velocities. Thus oneions integrated over time to

er a series of [A], whereby eacho ra e cons an s. us e

rmined. Fitting the data

ces the rigor of the fits.

r such a global fit is a strongs theoretically appropriate.


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Notes:Rate constants are over-determined,

. . 1 -1 from association reaction; k-1 can bedetermined independently fromdissociation. Moreover, in a series

,should be consistent.

From the association rxn, k-1 is bestdetermined when k1A k-1, i.e. atow . rom ssoc at on rxn, -1

is best determined when [AB] is large,i.e. at high [A].

In general, the best strategy is touse global curve fitting of integratedrate e uations coverin both

association and dissociation reactions(BIAEVAL3 or CLAMP).


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Notes:

As rxn 1 becomes rate-limitin , formation of[AB] becomes zero-orderwrt [B].Large [B]0 favors rate-

limitation by rxn 1.

Rate of dissociation isnot validly described bysimple, exponentialdecay, because [A]t 0for all t.

Rnx 1 is a complexfunction of flow-rate,ligand size and Fick

diffusion coefficient,sensor geometry, etc.;thus the meaning of k1and k-1 is not easilyinterpreted.

Fitting global, integrated rate equati(simple bimolecular mechanism using

nBIAcores BIAevaluation software)


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(simple bimolecular mechanism, using

Note bulk-phase refra

BIAcore s BIAevaluation software )

tive index shifts


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BIAcore xa le: se ue

protein-protein interacti

Biological background: smallpox vimmune mo u a on pro e ns, w

response to the virus. Among thesbinding protein, which specifically

,

Experimental design: 44 residues o

Purified vaccinia virus chemokine-bas mobile phase, and the Kd measu

ce de endence o

on

ruses make a variety ofn er ere w os mmune

is a 35 kDa chemokineinds monocyte chemo-

, .

f MCP-1 were mutated,

inding protein was perfuseded.

BIAcore study of smallpox viruschemokine inhibitors

.T. Seet et al.,

NAS 98:9008 (2001)


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chemokine inhibitors

This study looked at affinity of 44 mutants ofrotein monoc te chemoattractant rotein

immobilized) towards the vaccinia chemokinKda; mobile phase ligand). Nine of the amintested markedly decreased affinity (i.e. increa

NAS 98:9008 (2001)

human MCP-1 77 residues

-binding protein (35acid substitutions

sed Kd).


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Another BIAcore exam le: cblood-coagulation factor bind

Biological background: Zymogens of t

activated ex osin domains rich in thtranslationally modified residue, -carfactors require Ca2+ bind to phospholisalt bridges between -carboxyglutam

Experimental design: 25 % phosphotivesicles (~100 nm ) are prepared andme o w c r ges rom pro e n oSince FXa binding is calcium depende

bound protein by washing with EDTA.

nstructin vesicles to measureng

he blood-clotting cascade are

vitamin K-de endent ost-boxyglutamic acid. These activatedid vesicles, presumably by formingte and phosphate head-groups.

ylserine + 75 % phosphotidylcholineimmobilized on Au-chips in a clever

o ny a e ags o p ayer.t, the vesicles are easily stripped of

Construction of immobilize vesicles:


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Construction of immobilize vesicles:A. Wikstrm and J.

Deinum;

Probing the interaction

of coagulation factors

with phospholipid vesicle

surfaces by surface

plasmon resonance,nalyt. Biochem. 362: 98

(2007)

25% phosphatidylserine:

+ defined number of A (dsDNA)per vesicle (145 nm, preparedeitherin vacuoor under N2)

biotinylated DNA complementaryto the sin le-strand tail of A

adsorbed, biotinylated BSA

neutravidin

bare Au chip

1 DNA / vesicle 12 DNA / vesicle


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Mobile phasesare coalgulation

factor FXaat 400, 200,100,50,25,and12.5 nM

plus 10 mM Ca2+

phospholipidsdried in vacuo

phospholipidsdried in N


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dependent, the vesicles could becleanly stripped of bound FXa,

without destroying their bindingcapacity, by a pulse of EDTA.

quilibrium constants were independent


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quilibrium constants were independentoa ng, w e assoc a on ra e

onstants decreasedsignificantly withNA loading (implying that dissociationate constants increased.


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Comparison of the binding of several fa,content and binding equilibrium.

tors show that Gla (-carboxylglutamic


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BIAcore SPR instrument

s


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1. We have considered BIAcore as athe binding reaction can be obsersignal. The total column volume

.but may be a complex, biological mcomponents that bind to the immo

uantified b mass .

Q: Is there any way to identify qualitatie

A: Not by SPR, but the mass captured

mass s ectrometr ! Indeed three

-- elution of the entire captured co-- on-chip proteolysis, with subseq

spray-ionization ms-- removal of the chip to use as a M

ini-affinity chromatograph, in whiched directly as the SPR optical

is on the order of 1 l.

,ixture, from which someilized phase will be captured, and

vely what these components might

is sometimes sufficient to identify by

roaches have been successful:

ponentent elution of peptides into electro-

LDI-TOF sample target

Coupling SPR to mass spectro eters: elution


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p g p

This approach simplyinjects an elution-

between air bubbles --across the chip

,the eluant.

In standard anal ticalruns effluent from thereaction cell goes towaste. But the streamcan e verte acinto the instruments

tube rack.The collected eluant isthen processed off-line

-

MALDI-TOF MS.

Coupling SPR to mass spectrousing the chip as MALDI targ

eters:t


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using the chip as MALDI targ tIn this mode, one physically

removes the chip from theinstrument and separates itrom e carr er.

Trypsinization insitu is

.

Matrix is added directly to the

The chip is then placed directlyon a MALDI-TOF-MS target.

The chip can be used only once,

and requires an especiallymac ne arge o r ng echip surface to the laser focalplane, with electrical connection

.

Interfacing BIAcore to ESIon-chi di estion

-MS-MS:


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on chi di estion

Natsume, Nakayama

and Isobe, A TRENDS

Guide to Proteomics, a

Biotechnology, 19:S28(2001).

A thirda roach is topark a trypsinsolution in the

cell to hydrolizethe boundprotein in situ.

e re ease

peptides arethen routed

nano-LC-MS-MS, for de

BIAcore SPR: summar


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BIAcore SPR summar

1. Surface plasmon resonance is a

2. It is sufficiently sensitive to detecis solute mass caused by a bind

3. The BIAcore implementation printerface to SPR, with a variety of

.

4. Monitoring binding in real time aloff-kinetics as well as equilibria.

5. Depending on experimental desiprotein:protein interactions.

6. Experimental problems are potenimmobilization geometry artifa

ptical phenomenon that depends of

t an increase in n due to an increaseing event.

ovides a miniaturized affinity columnbiochemically friendly surfaces

ows direct measurement of on- and

n, it detects ligand:protein or

ial diffusion limited kinetics andts.


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MALS: basic concepts


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. nsp re y e ques on y s

3rd Baron Rayleigh (a.k.a Lord Ratheoretical description of light sca.

2. Maxwell had just enunciated the th

based on the idea that the electricwithin a molecule, and consequenand tra ector .

3. The pattern of trajectory change, tof angular deviation from the unscthe radius of gyration of the molecmolecule, as with globular protein

molecular size and mass.

4. For mixtures of molecules, where ttumble independently, scattering i

interfering.

e s y ue , o n am ru ,

leigh) in 1871 developed atering. We still refer to Rayleigh

eory that light waves were composed

.vector of light could polarize chargesly itself undergo a change in phase

at is, the light intensity as a functionattered direction, can be related toule. Assuming a roughly spherical, this radius can be calculated as

he molecules are not associated andsimply additive. Where molecules

,

MALS: basic concepts (con

t)


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5. For particles whose radius of gyrathe light (e.g. globular proteins le

scattering is said to be isotropic.anisotropic, and yields information

6. Because it registers size and massdetermining the aggregation statehetero-complexes.

7. The method works with static samcoupled with size-exclusion chroma

ion is smaller than the wavelength ofs than about 5x106 Da), the angular

For larger particles the scattering ison the shape of the particle

the method is useful forf homopolymeric proteins as well as

les, but is particularly useful whenography in a constant-flow mode.

MALS: instrument designhe 18 diodes measure light


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intensity at their corresponding

ngles continuously andimultaneously, and with aens v y a ma es poss e oonitor a flow-cell (with volume =

.5 l) or much larger static

.

For small, isotropicalyscatering proteins, the


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term (virial coefficient

becomes negligible, inwhich case, K* simply

relating scatteringintensity to MW.

e cons an s reproportional to (dn/dc)2

which measuresolarizabilit of the mol

and inversely proporti

to 4.

For isotropic scatterers2nd and higher terms ofare sma , an usua y

ignored.


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Traditionally, the Zimm plot is used toanalyze MALS data. In effect, this takesdata in two inde endent variables, c and


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, and plots them in a single 2Dcoordinate system.

The k term is a gimmick to bring kc ansin2(/2) into the same numerical range.

A2 from initial

slope

Linearized plots likeZimms were popular in a

re-com uter a e. Neither

c nor are continuous, andthe approach is still widelyused.

cK 2 d R 2

.

rms radius from initialslope

The two dependencies of the Zimm plottheoretical components of scattering. Tintramolecular scatter; the linear relatio

on c and ) correspond to twoe linear relationship with c reflectsshi with reflects intermolecular


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scattering.

cK 1

MR 0 2

Intramolecular scattering arises when aphoton interacts with several parts ofthe same molecule before it is detected.The ideal case, where there is no

intramolecular scatter, corresponds tolow angles,,where as 0, .For practical reasons R(0) cannot be

measured directly!

A2 from i

slope

ncK 116 222

0

2

MMR g 23 20

Intermolecularscattering arises whena photon scattered from one molecule

is detected. The ideal case, wherethere is no intermolecularscatter,corres onds to low concentrations, i.e.where c 0

itial

rms radius frominitial slope

A Zimm plot of bovine serum albumin monomer:


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1

c1

c

Molar Mass (MM) : (7.7140.01)e+4 g/mol (0.16%RMS Radius (Rz) : 2.62.2 nm (2nd virial coefficient : (1.4130.06)e-4 mol mL/g2 (

source: Wyatt Technologies

c52 3

c3

c4

The curvature as c increases

2represents some non-ideality, probably significantdimer formation.

)84%)3%)

Note that molecular mass isdetermined with high precision,comparable to ms.

But the error in radius of gyration islarge, as a consequence of the factthat BSA is an isotropic scatterer.

MALS is most useful for proteinsize-exclusion chromatography (

in conjunction withSEC)

Degasser: MALS is verysensitive to out gassing!!


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sensitive to out-gassing!!

Filter: MALS is verysensitive to large particles(e.g. dirt)!!

UV detector: with proteinsthe UV signal can used tosupply the neededconcentration term (seetheory); refractive indexdetector is less useful, butcan e use o - ine to

establish dn/dc.DAWN-EOS: is the MALSetector. or e uent

protein peaks, the MALSsignal can be computed to

accurate (a few %) MWsw t out ca rat on.

Since SEC chromatography ca-

measure MW, why couple it


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-.prior calibration. (See next slide.)

2. Static samples of mixed aggregatbecause they yield weight-averagMALS can quantify ratios.

. ,standard UV trace, but calculatesacross a peak, revealing inhomog

Note that, in principle, any chromato,

index is a big problem.

Note also that proteins that absorb a(unless dn/dc is known) present

,

on states are difficult to interpretMW; by separating MW classes,

MW

at several concentrationseneities and aggregations.

graphic process can be linked to

t 633 nm, and glycoproteinsroblems.

MALS-SEC: molecular weight a curacy


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,

E. L Folta-Stogniew and K.R. Williams, J.

=, , , .

iomolec.Techniques, 10:51 (1999)

MALS-SEC examples: molecular

e ye p o s s m ar o a mm

weight of ovalbumin

o , u gnores e 2c erm an


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y pinverts the ordinate, that is, R()/

ovalbumin (monom

E. L Folta-Stogniew and K.R. Williams, J.

, g 2*c

This plotcorresponds to the

r), sequence MW = 42.8 kDa

single slice atthe apex of thechromatographicpeak. The root-mean-square-

radius,1/2

, isthe slope; MW isthe intercept.

The AUX detector

a UV detector, andsupplies c (on aslice-by-

.

iomolec.Techniques, 10:51 (1999)

MALS-SEC: more examples E. L. Folta-Stogniew and K.R. Williams,J.Biomolec.Techniques, 10:51 (1999)


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In this representation of the data,the Debye-intercept (= MW) for eachslice of the chromatogram issuperimposed on thechromatographic trace as a

.

=46.7 kDa; molecular calibration ofthis SEC column gives an MW ~ 50

kDa. But MALS calculates a Mindicating the enolase dimer!!

One expects the 93.3 kDa to elute before.

The authors explain it this way:

i h f h SEC l

,i b h h


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penetrating the pores of the SEC-colu

sequestering monomer from participati-

equilibrium, and exists mostly as dime

the dimer should elu

n, it behaves as a monomer, thus

on in the equilibrium. When emerging-

!

te about here

SaDebye plots

C-MALS: monitoring thesembly of virus capsids:p149 is an expressed version of the


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HC

y psuperimposed

on SEC

Protein and saltdependence ofassembly

p1492 is an expressed version of the

B capsid protein lacking a 34-residue,terminal, RNA-binding domain.

4 x 106 Da / 3.5 x 104 Da => 114.3dimers in capsid. CyroEM imagessugges an cosa e ron. us slikely that there are 60 tetramers inT=4 pseudosymmetric icosahedral

. , -

Protein Interactions Are Sufficient toDrive Assembly of Hepatitis B Virus

Capsids, Biochemistry 41:11525 (2002)

Sa

Calculatin

C-MALS: monitoring thesembly of virus capsids: (cont)

G from MALS data and H from


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Calculatin

corresponthermodye uce .

molecular

-but the nis, apparefavorable

ln

P.

Pr

Dr

G from MALS data and H froming vant Hoff plots, a completeamic description of the process is

Calculate values are erive rommodels.)

,gative entropy attendant on organizationntly, more than offset by the entropicallyburial of hydrophobic interfaces.

Keq H0

R1T

S

0

R

Ceres and A Zlotnick, Weak Protein-

otein Interactions Are Sufficient to

ive Assembly of Hepatitis B Virus

sing MALS to measure


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So

rotein:This 16 kDa protein contains just 8potential sites for PEGylation.

Hence extend of PEG lation shouldbe between 16 kDa (0 PEGs) and 56kDa (+ 8 5 kDa).

In fact, the RI trace shows a small

impurity, which 90-scatter traceshows to be very high MW.

Slice-by-slice Debye analysisindicates a large aggregate. Such

FDA) because of theirimmunogenicity.

urce: Wyatt Technology, Applications Librar

Dimerization Equilibrium and Fuodulator Protein:

ctionality of a Viral Immune

t ti l l h th t th


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Vaccina virus encodes aprotein (B14) which blocks h

-activation of NF-B. B14 existsin monomer:dimer equilibrium,

Ia

. d

Benfield et al., Mapping the IB Kinase (IKK)- bindi

Inhibitor of IKK-mediated Activaton of Nuclear Facto

utational analyses shows that theydrophobic face of the monomer

K complex to prevent NF-Bctivation. That is, that the IKK

imerization.

ng Interface of the B14 Protein, a Vaccinia Virus

B; JBC 285:20727 (2011)

DimeriFunctiModul

ation Equilibrium andnality of a Viral Immune

tor Protein: (cont)


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A.

B.

B.

Wit

( )

Shows the dimerization interface(helices 1 and 6) of w.t. B14

& C. show the mutationalreplacement of four hydrophobicresidues with hydrophilic ones.

Shows the results of MALS analyses

indicating that 3 of these mutationsprevented dimerization entirely,while T31K weakened dimerization.

the exception of Y35E, disruptionof this hydrophobic interaction face

mutants to trigger NF-B signaling.

Multi-Angle Light Scattering: Summary


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1. Rayleigh scattering of monochrommacromolecules yields informatiomolecular mass of the molecules.

2. Thus it is useful for studying the hof proteins in solution.

3. Because light scattering intensity iis particularly appropriate for char,

nanoparticles.

tic, plane-polarized light fromabout the shape, size and

mo- and hetero-oligomerization

creases with molecular size, itcterizing large aggregates such, ,

prot interaction

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