Oxometalates as artificial proteases: sequence versus region selectivity
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Oxometalates as artificial proteases: sequence versus region selectivity
Karen Stroobants
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Oxometalates as artificial proteases: sequence versus region selectivity
Inorganic chemistry
Protein chemistry
« Why is protein hydrolysis important? »
« What is wrong with trypsin? »
- limited solvent compatibility- unreactive toward IDPs- small fragments
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Often proposed solution:
The use of artificial metallopeptidases
metal ions as hydrolytic active centers
Current problems:
Low reactivity at physiological pH and temperature
Non specific interaction / Non selective cleavage
“The development of alternative cleavage strategies would greatly facilitate the study of protein structure and function.”
Grant 2006
Need for complementary and versatile cleavage agents
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Hetero Polyoxometalates“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”
“A multitechnique study of europium decatungstate and human serum albumin molecular interaction”
(Nadjo et al.)
Conceptually new artificial cleavage agents
Iso Oxometalates
“Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, 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.)
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Hetero Polyoxometalates“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”
“A multitechnique study of europium decatungstate and human serum albumin molecular interaction”
(Nadjo et al.)
Conceptually new artificial cleavage agents
Iso Oxometalates
“Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, 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.)
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Molybdate(IV) hydrolyzes HEWL
SDS-PAGE &Edman degradation:
Asp18-Asn19Asp48-Gly49Asp52-Trp53Asp101-Gly102
pH 5.0 / 60 °C
+Ho et al. (2011) Inorg. Chem.Stroobants et al. (2013) submitted to J. Inorg. Biochem.
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Sequence specific hydrolysis at Asp-X bonds
Interaction occurs at many positions and thus is not selective
Reaction only occurs when an Asp-X bond is accesible and thus is sequence selective
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Side chain assistence mechanism
Polarization of the carbonyl due to [MoO4]2- binding & Internal nucleophilic attack of the Asp side chain
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Side chain assistence mechanism
Polarization of the carbonyl due to [MoO4]2- binding & Internal nucleophilic attack of the Asp side chain
Appearance of Gly
Disappearance of Asp-Gly
ppm
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Hetero Polyoxometalates“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”
“A multitechnique study of europium decatungstate and human serum albumin molecular interaction”
(Nadjo et al.)
Conceptually new artificial cleavage agents
Iso Oxometalates
“Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, 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.)
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Polyoxotungstate ligand
SPECIFIC INTERACTION
Polyoxotungstates as more tunable alternative
Lewis active metal
HYDROLYTIC ACTIVITY
- Higher stability (vs. Oxomolybdate)- Known region specific interaction with protein surfaces- Not reactive as such!!!
RC
NR
O
H
OH
- Oxophilicity- Coordination number- Ligand exchange kinetics Ce(IV) ion
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Zr(IV)-substituted POMs hydrolyze HSA
…KQNCys392//Glu393LFE…
…VESLys313//Asp314VCK…
…DDRAla257//Asp258LAK…
…NLPArg114//Leu115VRP…
Stroobants et al. (2013) Chem. Eur. J.
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lacunary Wells Dawson
Zr(IV) substitutedWells Dawson
1:1 interaction / 1:3 interactionKa ~ 108 M-1/ Ka ~ 105 M-1
1:1 interactionKa ~ 106 M-1
Zr(IV) and Wells Dawson POM driven interactions
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* Arg114-Leu115
known POM binding positive regionNadjo et al.
* Ala257-Asp258* Lys313-Asp314* Cys392-Glu393
regions of mixed charge, hydrolysis downstream from acidic residue
Zr(IV)-Wells Dawson exhibits region selective interaction
lacunary Wells Dawson
Zr(IV) substitutedWells Dawson
1:1 interactionKa ~ 106 M-1 1:1 interaction / 1:3 interaction
Ka ~ 108 M-1/ Ka ~ 105 M-1
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Additional fragments upon prolonged reaction time
…VESLys313//Asp314VCK…
…DDRAla257//Asp258LAK…
Edman degradation
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Same selectivity for secondary fragmentation
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Same selectivity for secondary fragmentation
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Reaction occurs- at pH 7.4- at Asp and Gluresidues
* Ala257-Asp258* Lys313-Asp314* Cys392-Glu393
regions of mixed charge, hydrolysis downstream from acidic residue
Is X-Asp/Glu hydrolysis mechanistically related to Asp-X cleavage?
Proposed assistence mechanism- does only dominate < pH 6- does only occur for Asp(not for Glu)
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Different mechanisms for oxometalate versus metal-substituted POM reactivity
Non-specific binding
Sequence selective hydrolysis,via side chain assistence mechanism
Region selective interaction
Hydrolysis without side chain assistance, presumably via external water nucleophile delivery
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(Poly)oxoanions hold promise as artificial proteases
Evidence for * Sequence selective Asp-X protein hydrolysis in the presence of oxomolybdate & * Region selective protein cleavage in the presence of metal-substituted polyoxotungstates
Future perspective
« Hydrolysis of membrane proteins and IDPs possible? »
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Many thanks to…
My promotor – Prof. Parac-Vogt Tatjana
POM post-doc – Dr. Absillis Gregory
Edman degradation – Prof. Proost Paul & Dr. Moelants Eva
ITC – Dr. Bruylants Gilles
NMR technician – Karel Deurinckx
Master students – Laure Baeyens, Dounia Saadallah, Anja Bourgois, Vincent Goovaerts & Jeroen Lannoeye
All the colleagues of lab LBC / LIC / ULB
FWO for financial support
& you for your attention!