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Oxometalates as artificial proteases: sequence versus region selectivity. Karen Stroobants. Inorganic chemistry. «  Why is protein hydrolysis important? ». Oxometalates as artificial proteases: sequence versus region selectivity. «  What is wrong with trypsin ? ».

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slide1

Oxometalates as artificial proteases:

  • sequence versus region selectivity

Karen Stroobants

slide2

Inorganicchemistry

« Whyisproteinhydrolysis important? »

  • Oxometalates as artificial proteases:
  • sequence versus region selectivity

« Whatiswrongwithtrypsin? »

  • limitedsolvent compatibility
  • unreactivetowardIDPs
  • small fragments

Proteinchemistry

slide3

Need for complementary and versatile cleavage agents

“The development of alternativecleavage strategies would greatly facilitate the study of protein structure and function.” Grant 2006

Oftenproposed solution: The use of artificialmetallopeptidases

 metal ions as hydrolytic active centers

Currentproblems:

 Lowreactivityatphysiological pH and temperature

 Non specific interaction / Non selectivecleavage

slide4

Conceptuallynewartificialcleavageagents

IsoOxometalates

“Sarcoplasmicreticulum calcium ATPaseinteractionswithdecaniobate, decavanadate, vanadate, tungstate and molybdate”

“Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.)

Hetero Polyoxometalates

“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”“A multitechnique study of europium decatungstateand human serum albuminmolecularinteraction” (Nadjo et al.)

slide5

Conceptuallynewartificialcleavageagents

IsoOxometalates

“Sarcoplasmicreticulum calcium ATPaseinteractionswithdecaniobate, decavanadate, vanadate, tungstate and molybdate”

“Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.)

Hetero Polyoxometalates

“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”“A multitechnique study of europium decatungstateand human serum albuminmolecularinteraction” (Nadjo et al.)

slide6

Molybdate(IV) hydrolyzes HEWL

SDS-PAGE &

Edmandegradation:

Asp18-Asn19

Asp48-Gly49

Asp52-Trp53

Asp101-Gly102

pH 5.0 / 60 °C

+

Ho et al. (2011) Inorg. Chem.

Stroobants et al. (2013) submitted to J. Inorg. Biochem.

slide7

Sequencespecifichydrolysis at Asp-Xbonds

Interactionoccurs at manypositions and thus is notselective

ReactiononlyoccurswhenanAsp-X bond is accesible and thus is sequenceselective

slide8

Side chainassistencemechanism

Polarization of the carbonyl due to [MoO4]2- binding &

Internal nucleophilic attack of the Asp side chain

slide9

Side chainassistencemechanism

Appearanceof Gly

Disappearanceof Asp-Gly

Polarization of the carbonyl due to [MoO4]2- binding

&

Internal nucleophilic attack of the Asp side chain

ppm

slide10

Conceptuallynewartificialcleavageagents

IsoOxometalates

“Sarcoplasmicreticulum calcium ATPaseinteractionswithdecaniobate, decavanadate, vanadate, tungstate and molybdate”

“Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.)

Hetero Polyoxometalates

“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”“A multitechnique study of europium decatungstateand human serum albuminmolecularinteraction” (Nadjo et al.)

slide11

Polyoxotungstates as more tunablealternative

Polyoxotungstateligand

SPECIFIC INTERACTION

Lewis active metal

HYDROLYTIC ACTIVITY

  • Higherstability (vs. Oxomolybdate)
  • Knownregionspecificinteractionwithprotein surfaces
  • Notreactive as such!!!
  • Oxophilicity
  • Coordinationnumber- Ligand exchange kinetics
  •  Ce(IV) ion
slide12

Zr(IV)-substitutedPOMshydrolyze HSA

…NLPArg114//Leu115VRP…

…DDRAla257//Asp258LAK…

…KQNCys392//Glu393LFE…

Stroobants et al.

(2013) Chem. Eur. J.

…VESLys313//Asp314VCK…

slide13

Zr(IV) and Wells Dawson POM driveninteractions

lacunaryWells Dawson

Zr(IV) substitutedWells Dawson

1:1 interaction

Ka~ 106M-1

1:1 interaction / 1:3 interaction

Ka~ 108M-1/ Ka ~ 105M-1

slide14

Zr(IV)-Wells Dawson exhibitsregionselectiveinteraction

lacunaryWells Dawson

Zr(IV) substitutedWells Dawson

1:1 interaction

Ka~ 106M-1

1:1 interaction / 1:3 interaction

Ka~ 108M-1/ Ka ~ 105M-1

* Arg114-Leu115

known POM binding positiveregionNadjo et al.

* Ala257-Asp258

* Lys313-Asp314

* Cys392-Glu393

regions of mixed charge, hydrolysis downstream fromacidicresidue

slide15

Additionalfragmentsuponprolongedreaction time

Edmandegradation

…DDRAla257//Asp258LAK…

…VESLys313//Asp314VCK…

slide18

Is X-Asp/Gluhydrolysismechanisticallyrelatedto Asp-Xcleavage?

Proposedassistencemechanism

- does onlydominate < pH 6

- does onlyoccurforAsp

(notforGlu)

* Ala257-Asp258

* Lys313-Asp314

* Cys392-Glu393

regions of mixed charge, hydrolysis downstream fromacidicresidue

  • Reactionoccurs
  • at pH 7.4
  • at Asp and Glu
  • residues
slide19

Different mechanismsforoxometalate versus metal-substituted POM reactivity

Non-specific binding

Sequenceselectivehydrolysis,

via sidechainassistencemechanism

Regionselectiveinteraction

Hydrolysis without sidechainassistance, presumably via external

water nucleophiledelivery

slide20

(Poly)oxoanionsholdpromise as artificialproteases

Evidencefor* SequenceselectiveAsp-Xproteinhydrolysis in the presence of

oxomolybdate

&

* Regionselectiveproteincleavage in the presence of metal-substitutedpolyoxotungstates

Futureperspective

« Hydrolysis of membrane proteins and IDPs possible? »

slide21

Manythanks to…

My promotor – Prof. Parac-Vogt Tatjana

POM post-doc – Dr. AbsillisGregory

Edmandegradation – Prof. Proost Paul & Dr. Moelants Eva

ITC – Dr. Bruylants Gilles

NMR technician – Karel Deurinckx

Masterstudents – LaureBaeyens, DouniaSaadallah, Anja Bourgois,

Vincent Goovaerts & Jeroen Lannoeye

All the colleagues of lab LBC / LIC / ULB

FWO forfinancial support

& youforyourattention!