isothermal titration calorimetry for bioinorganic chemists
TRANSCRIPT
Isothermal Titration Calorimetry for Bioinorganic Chemists:
Technical Issues, and Applications for New Insight�
�Dean Wilcox �
�Department of Chemistry�
Dartmouth UniversityHanover, NH �
College
Outline – 2014 Penn State WorkshopCalorimetryBiomolecular Calorimetry Isothermal Titration Calorimetry (ITC)
The Method: information, instruments, data, analysisBioinorganic Applications Issues with metal ions His (Ni2+), a teaching example Examples: Transferrin (Fe3+) Ferritin (Fe2+) Albumin (Cu2+) His-rich sequence (Zn2+, Fe3+, Fe2+, etc.) Metallothionein (Zn2+, Cd2+, As3+, Pb2+) UreE, a urease metallochaperone (Ni2+, Cu2+)
Differential Scanning Calorimetry (DSC)The Method: information, instruments, dataBioinorganic Applications: Insulin (Zn2+), Carbonic Anhydrase (Zn2+)
Outline – 2016 Penn State Workshop
Isothermal Titration Calorimetry (ITC)The Method: information, instruments, data, analysisTechnical IssuesBioinorganic Chemistry Applications Issues with Metal Ions Metal-buffer interaction Examples: Azurin (Cu2+, Cu+) Metallothionein-3 (Pb2+, Zn2+) Ln3+-binding peptides (Dy3+, Nd3+)
Thermodynamics of Metal Ions Binding to Proteins
Mn+ + Protein Mn+Protein
- Equilibrium (K) K = [Mn+Protein] / [Mn+][Protein]
- Free Energy (ΔG) ΔG° = -R T ln(K)
- Enthalpy (ΔH) calorimetry: ΔH° = qP
- Entropy (ΔS) ΔG° = ΔH° - TΔS°
- Heat Capacity (CP) ΔCP = ΔH°0 / T0
- ΔH° / T = ΔΔH° / ΔT
→←
Isothermal Titration Calorimetry
Isothermal Titration CalorimetryTime
Molar Ratio [Titrant/Cell]
kcal/mol of injectant
µcal/sec
Binding Isotherm
Raw Data
Capabilities - determine binding stoichiometry and K value (3 < log KITC < 8). - complete thermodynamic profile in one experiment (ΔG°, ΔH, ΔS). - determine ΔCp from temperature dependence of ΔH.
c = K [P]
ITC: Uses
1. Basic Is there binding? What is the binding stoichiometry (n)? Caveat: generally softer number What is the binding affinity (Kd)? Caveat: c window limitations
2. Intermediate What is the binding enthalpy (ΔH)? Caveat: measuring net ΔHITC Is there coupled (de)protonation that accompanies binding (nH+)? What is the binding entropy (ΔS)? Caveat: from comparable ΔG, ΔH
3. Advanced What is the change in heat capacity (ΔCp)? What are the effects of other perturbations (ionic strength, solvent, etc.)? Kinetics of binding?
ITC: What to do with the data?1. Manipulation of the data.
- baseline correction- subtract heat of dilution control titration extended region of experimental data-integration
2. Qualitative analysis of the data (critical evaluation).injection peaks? stoichiometry? c-window? enough heat? (concentration, conditions)(initial data à better data à optimal data à reproducible data)
3. Quantitative analysis of the data. a) Fitting to a model
- Single binding site model à n, K, ΔH - Independent binding sites model (2) - Sequential binding sites model - Competition binding model à known KA, ΔHA; unknown KB, ΔHB
b) Post-hoc analysis of KITC and ΔHITC values (condition-dependent values à “condition-independent” values)
ITC: Technical Issues
Red Flags Noisy data?: precipitate, air bubbles Irreproducible results?: cleanliness, air bubbles, chemistry (!) Low net heat (ΔHITC)?: use another temperature, other conditions (buffer) Slow kinetics (return to baseline)?: faster stirring, higher temperatureSources of Error Binding stoichiometry (n): depends on two concentrations and volumes Binding enthalpy (ΔH): measuring net heat flow (post-hoc analysis) Binding constant (K): measuring overall equilibrium (post-hoc analysis)
competing equilibria compare/confirm with other method(s)
ITC of Metal-Protein Interactions: Complications1. Metal-Buffer Interactions *
M(buffer)x+n M+n + X buffer
2. Metal Redox Reactions
M+n + [O] M+(n+1) + [R]
3. Metal Solution Chemistrya) Hydrolysis
LnM+n-H2O LnM+n-OH- + H+b) Precipitation / Dissolution
LnM(aq) LnM(s)4. Proton Displacement *
M+n + Protein M+nProtein + n H+n H+ + n buffer n bufferH+
→←
→←
→←
→←
→←
→←
ITC of Metal-Protein Interactions: Experimental Design1. pH.- metal ions are Lewis acids and compete with H+- coupled equilibria involving H+ are common with metal ions- need to account for heat associated with coupled (de)protonations
2. Buffer.a) interaction with the metal ion - can suppress side reactions of the metal ion (e.g., hydrolysis) - can provide a competing ligand for the metal ion - need to know KM-buff and ΔH°M-buffb) interaction with H+ - ΔH°buff-H+ can be used to quantify H+’s in coupled equilibria - use a buffer with larger heat of protonation to amplify signal
3. Other species?- reducing agent, complexing agent, salt, detergent, DMSO, etc.
Overall: (1-z)M2+ + zMB2+ + (1-x)L + xHL+ + (x- z)B ML2+ + xHB+ ���Buffer- and pH-Independent Equilibrium: M2+ + L ML2+
(1-z)M2+ + zMB2+ + (1-x)L + xHL+ + (x- z)B ML2+ + xHB+
ΔHITC
MB2+ M2+ + B z -ΔHMB
HL+ H+ + L x -ΔHHL
H+ + B HB+ x ΔHHB
M2+ + L ML ΔHML
→←
→←
→←
→←
→←
Thermodynamic cycles are required to obtain condition-independent values from condition-dependent experimental values
→←
→←
Penn State Workshop Tutorial:�Cu2+ à albumin
Lys-4
Most abundant circulatory protein (~ 0.6 mM).
Binds and transports Cu+2 and Ni+2 at
its N-terminal X-X-His- binding site (not resolved in structure).
Human Serum Albumin (PDB file 1UOR(HSA))
ß C
N
O
CH
H2N
N
M
N
HN
CH2
C
CH
CH
CONHR
O
2+
(-)
R1
(-)
R2
Cu2+ Binding to AlbuminCu+2 binding to bovine serum albumin (BSA)(reported, pH-independent, and pH 7.4 equilibrium constants
Method pH Buffer Competing
Ligand
log Kreporteda log Kcalc..
b log KpH 7.4 c Reference
Ultrafiltration 7.5 c MOPS
(50 mM)
Gly 13.2 -1.26 13.0 Giroux and
Schoun, 1981
Dialysis 7.4 12.04 -2.12 12.2 Ryall
1974
7.0 d HEPES
(30 mM)
His 11.12 -1.79 12.5 Saltman,
et al, 1993
8.5 d HEPPS
(30 mM)
His 11.12 -5.80 8.5 Saltman,
et al, 1993
Ion-selective
electrode
7.3 HEPES
(20 mM)
13.2 -0.67 13.6 Ljones,
et al, 1986
7.3 BisTris
(46 mM)
12.6 -1.27 13.0 Ljones,
et al, 1986
5.9 Acetate
(25 mM)
11.2 2.36 16.7 Ljones,
et al, 1986
a) Kreported = [Cu(BSA)]/[Cu2+][BSA]; reported apparent binding constant at the given pH
and experimental conditions.
b) Kcalc.. = [Cu(BSA)][H+]2/[Cu2+][BSA]; intrinsic equilibrium constant for the reaction:
Cu2+ + BSA →← Cu(BSA) + 2H+
c) KpH 7.4 = [Cu(BSA)]/[Cu2+][BSA]; apparent binding constant at pH 7.4.
• Use competition with buffer (e.g. Tris) to measure high affinity Cu2+ binding.
Cu2+ Binding to Albumin
Cu2+ à BSA
100 mM Tris pH 9.2(100 mM borate, 20 mM NaCl)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-10.00
-8.00
-6.00
-4.00
-2.00
0.00-4.00
-3.00
-2.00
-1.00
0.00
0 60 120 180 240
Time (min)
µcal/
sec
[CuSO4]/[DTPA]
kcal
mol
-1 o
f inj
ecta
nt
16
0.0 0.5 1.0 1.5 2.0 2.5
-6.00
-4.00
-2.00
0.00
-2.50
-2.00
-1.50
-1.00
-0.50
0.00
0 30 60 90 120 150
Time (min)
µcal
/sec
[CuSO4]/[TEPA]
kcal
mol-1
of i
njec
tant
1.0 mM TEPA à 0.1 mM CuSO4100 mM bis-Tris, 100 mM NaCl
1.2 mM DTPAà 0.1 mM CuSO4100 mM bis-Tris, 100 mM NaCl
Cu2+-buffer interaction
Cu2+-buffer interaction (pH 7.0)
17
TEPA DTPAAverage of Both
Ligands
Buffer Log βnΔHMB
(kcal/mol) Log βnΔHMB
(kcal/mol) Log βnΔHMB
(kcal/mol)
bis-Tris 12.1 ± 0.4 –6.2 ± 0.7 11.8 ± 0.3 –7.9 ± 0.2 12.0 ± 0.3 –7.1 ± 0.4
TAPSO 13.0 ± 0.3 –11 ± 3 12.3 ± 0.5 –8 ± 3 12.7 ± 0.3 –9 ± 2
DIPSO 13.0 ± 0.3 –6.6 ± 0.4 10.8 ± 0.6 –5.8 ± 0.7 11.9 ± 0.3 –6.2 ± 0.4
TES 13.3 ± 0.3 –9.0 ± 0.7 13.2 ± 0.4 –9.3 ± 0.3 13.3 ± 0.3 –9.2 ± 0.4
Colette F. Quinn, Margaret C. Carpenter, Molly L. Croteau, Dean E. Wilcox“Isothermal Titration Calorimetry of Metal Ions Binding to Proteins”, in Methods in Enzymology, Vol. 567, Calorimetry, A. L. Feig, ed; Academic, 2016, pp. 3-21
ITC Studies of Metal Ions Binding to Proteins
1. Cu2+ and Cu+ binding to Azurin2. Pb2+ and Zn2+ binding to Metallothionein-33. Ln3+ binding to peptides
Azurin
19
Cu2+: Blue (PDB 4AZU)Cu+: Gray (PDB 1E5Y)Apo: Green (PDB 1E65)
74
Figure 4.2: (A) X-ray structure of Pseudomonas aeruginosa azurin (PDB: 4AZU) with metal-coordinating residues shown with bonds and titratable residues (His35 and His83) also shown for clarity. (B) Diagram of the Greek key protein fold of
azurin; (C) View of the copper metal site in azurin with coordinating ligands labelled, and the flexible loop between residues 112 and 121, shown in blue.
A
B C
1 2345
67
8
Cu2+ binding to Azurin: Blasie & Berg pH 7.0
20
0.0 0.5 1.0 1.5 2.0
-8.00
-6.00
-4.00
-2.00
0.00
-0.40
-0.30
-0.20
-0.10
0.00
0 60 120 180 240 300 360 420
Time (min)
µcal
/sec
[CuCl2]/[Azurin]
kcal
mol
-1 o
f inj
ecta
nt
1.0 mM apo-azurin à 0.028 mM CuCl2200 mM cholamine+, 50 mM NaCl
ΔH = –10.2 ± 1.6 kcal/molK = 4.0 x 1013
(log K = 13.6)
ΔS = –19.9 cal/mol K ΔSMetalDehydra.on
ΔSTransla.onalΔSConforma.onalBlasie, C.A.; Berg, J.M. JACS, 2003, 125, 6866-6867.
Dunitz, J.D. Science, 1994, 264, 670.Amzel, L. Proteins Struct. Funct. Bioinforma. 1997, 28, 144-149.
21
0 1 2 3 4 5
-1.00
-0.80
-0.60
-0.40
-0.20
0.00-0.25
-0.20
-0.15
-0.10
-0.05
0.00
0 30 60 90
Time (min)
µcal
/sec
[CuSO4]/[Azurin]
kcal
mol
-1 o
f inj
ecta
nt
Cu2+-Buffer ⇋ Cu2+ + Buffer –ΔHMBAzurin-Hm ⇋ Azurin + m H+ –ΔHHPCu2+ + Azurin ⇋ Cu2+-Azurin ΔHMP m Buffer + m H+ ⇋ m Buffer-H ΔHHB
ΔΗITC = ΔΗMP – ΔΗHP – ΔΗMB + m ΔΗHB
ΔΗMP – ΔΗHP = ΔΗITC + ΔΗMB – m ΔΗHB
1.0 mM CuSO4 à 0.03 mM apo-azurin100 mM bis-Tris, 100 mM NaCl
Perform experiment in > 3 buffers: y = mx + b
ΔHITC + ΔHMB = m ΔHHB + (ΔHMP – ΔHHP)
Cu2+ binding to Azurin pH 7.0
Cu2+ displacement of protons pH 7.0
22
Cu2+ displacement of protons pH 7.0
23
91
Scheme 4.6: Cartoon diagram of the theoretical proton flow occurring upon (A) Cu2+ or (B) Cu+ coordination to apo-azurin at pH 7.0, 25 °C. Determinations of bound protons at pH 7.0 are from Table 4.1.
Cu2+
His117
Cys112
His83His35
1.00
0.240.70
0.8
His117
Cys112
His83His35
1.00
0.240.76
0.8
Cu2+
0.06
Cu2+ Coordination1.80 ± 0.04 H+ displaced from metal site0.06 ± 0.14 H+ protonate His831.74 ± 0.15 H+ protonate the buffer
–
Cu+
His117
Cys112
His83His35
H+
0.690.84
H+
Cu+
0.140.45
0.8
1.00
0.28
Cu+ Coordination1.8 ± 0.04 H+ displaced from metal site0.45 ± 0.14 H+ protonate His350.14 ± 0.03 H+ protonate His830.28 ± 0.04 H+ protonate Me6T0.93 ± 0.15 H+ protonate the buffer
His117
Cys112
His83His35
1.00
0.240.70
0.8
A
B
–––
91
Scheme 4.6: Cartoon diagram of the theoretical proton flow occurring upon (A) Cu2+ or (B) Cu+ coordination to apo-azurin at pH 7.0, 25 °C. Determinations of bound protons at pH 7.0 are from Table 4.1.
Cu2+
His117
Cys112
His83His35
1.00
0.240.70
0.8
His117
Cys112
His83His35
1.00
0.240.76
0.8
Cu2+
0.06
Cu2+ Coordination1.80 ± 0.04 H+ displaced from metal site0.06 ± 0.14 H+ protonate His831.74 ± 0.15 H+ protonate the buffer
–
Cu+
His117
Cys112
His83His35
H+
0.690.84
H+
Cu+
0.140.45
0.8
1.00
0.28
Cu+ Coordination1.8 ± 0.04 H+ displaced from metal site0.45 ± 0.14 H+ protonate His350.14 ± 0.03 H+ protonate His830.28 ± 0.04 H+ protonate Me6T0.93 ± 0.15 H+ protonate the buffer
His117
Cys112
His83His35
1.00
0.240.70
0.8
A
B
–––
Experimental number of protons going to the buffer: 1.5 ± 0.3
Cu2+ binding to Azurin pH 7.0
24
Buffer n log K ΔG° (kcal/mol)
ΔH (kcal/mol)
–TΔS (kcal/mol)
ΔS (cal/mol K)
Cu2+
bis-Tris 1.0 ± 0.1 15.2 ± 0.3 –20.8 ± 0.4 0.8 ± 0.4 –21.6 ± 0.6 72 ± 2
TAPSO 1.0 ± 0.1 15.9 ± 0.4 –21.7 ± 0.5 2.8 ± 2.0 –24.5 ± 2.1 82 ± 7
DIPSO 1.00 14.9 ± 0.4 –20.4 ± 0.5 2.8 ± 0.5 –23.2 ± 0.7 78 ± 3
TES 1.00 15.6 ± 0.4 –21.3 ± 0.5 2.0 ± 1.0 –23.3 ± 1.1 78 ± 4
Average 15.4 ± 0.2 –21.1 ± 0.2 2 ± 1 –23 ± 1 78 ± 3
Enthalpically unfavorable• Coordination site is not tuned for Cu2+Entropically very favorable• Cu2+ desolvation; displaced protons; little rearrangement at site
Cu+ coordination chemistry
25
Prevent oxidation: anaerobic environmentSuppress disproportionation: Cu+-stabilizing ligand
2 Cu+(aq) ⇋ Cu2+
(aq) + Cu0
(s)
K = 1.3 (± 0.8) x 106
Me6Trien(Me6T)
1,1,4,7,10,10-hexamethyltriethylenetetraamine
pH 7.0CuIMe6TrienH1.69
2.69+log K = 13.6 ± 0.4
ΔHML = -7.4 ± 0.5 kcal/mol
0.0 0.5 1.0 1.5 2.0
-15.00
-12.00
-9.00
-6.00
-3.00
0.00
-1.50
-1.25
-1.00
-0.75
-0.50
-0.25
0.00
0 30 60 90 120 150 180
Time (min)
µcal
/sec
[Cu+]/[Azurin]
kcal
mol
-1 o
f inj
ecta
nt
Cu+ binding to Azurin pH 7.0
29
0.3 mM Cu+/16.5 mM Me6T à 0.03 mM apo-azurin/16.5 mM Me6T
100 mM HEPES, 100 mM NaCl
Cu+-Me6T ⇋ Cu+ + Me6T –ΔHMLMe6T + H+ ⇋ Me6T-H ΔHHLAzurin-H ⇋ Azurin + H+ –ΔHHPCu+ + Azurin ⇋ Cu+-Azurin ΔHMP m Buffer + m H+ ⇋ m Buffer-H ΔHHB
ΔHITC = ΔHMP – ΔHHP – ΔHML + ΔHHL + m ΔHHB
ΔHMP – ΔHHP = ΔHITC + ΔHML – ΔHHL – m ΔHHB
Perform experiment in > 3 buffers:y = mx + b
ΔHITC = m ΔHHB + (ΔHMP – ΔHHP + ΔHHL – ΔHML)
Cu+ displacement of protons pH 7.0
27
Cu+ displacement of protons pH 7.0
28
91
Scheme 4.6: Cartoon diagram of the theoretical proton flow occurring upon (A) Cu2+ or (B) Cu+ coordination to apo-azurin at pH 7.0, 25 °C. Determinations of bound protons at pH 7.0 are from Table 4.1.
Cu2+
His117
Cys112
His83His35
1.00
0.240.70
0.8
His117
Cys112
His83His35
1.00
0.240.76
0.8
Cu2+
0.06
Cu2+ Coordination1.80 ± 0.04 H+ displaced from metal site0.06 ± 0.14 H+ protonate His831.74 ± 0.15 H+ protonate the buffer
–
Cu+
His117
Cys112
His83His35
H+
0.690.84
H+
Cu+
0.140.45
0.8
1.00
0.28
Cu+ Coordination1.8 ± 0.04 H+ displaced from metal site0.45 ± 0.14 H+ protonate His350.14 ± 0.03 H+ protonate His830.28 ± 0.04 H+ protonate Me6T0.93 ± 0.15 H+ protonate the buffer
His117
Cys112
His83His35
1.00
0.240.70
0.8
A
B
–––
91
Scheme 4.6: Cartoon diagram of the theoretical proton flow occurring upon (A) Cu2+ or (B) Cu+ coordination to apo-azurin at pH 7.0, 25 °C. Determinations of bound protons at pH 7.0 are from Table 4.1.
Cu2+
His117
Cys112
His83His35
1.00
0.240.70
0.8
His117
Cys112
His83His35
1.00
0.240.76
0.8
Cu2+
0.06
Cu2+ Coordination1.80 ± 0.04 H+ displaced from metal site0.06 ± 0.14 H+ protonate His831.74 ± 0.15 H+ protonate the buffer
–
Cu+
His117
Cys112
His83His35
H+
0.690.84
H+
Cu+
0.140.45
0.8
1.00
0.28
Cu+ Coordination1.8 ± 0.04 H+ displaced from metal site0.45 ± 0.14 H+ protonate His350.14 ± 0.03 H+ protonate His830.28 ± 0.04 H+ protonate Me6T0.93 ± 0.15 H+ protonate the buffer
His117
Cys112
His83His35
1.00
0.240.70
0.8
A
B
–––
Experimental number of protons going to the buffer: 0.3 ± 0.1
Cu+ binding to Azurin pH 7.0
29
Buffer n log K ΔG° (kcal/mol)
ΔH (kcal/mol)
–TΔS (kcal/mol)
ΔS (cal/mol K)
Cu+
HEPES 0.8 ± 0.1 17.1 ± 0.5 –23.4 ± 0.7 –17.6 ± 1.0 –5.8 ± 1.2 20 ± 4
PIPES 0.8 ± 0.1 18.1 ± 0.6 –24.7 ± 0.9 –17.2 ± 1.3 –7.5 ± 1.6 25 ± 5
ACES 0.6 ± 0.1 17.9 ± 0.4 –24.5 ± 0.6 –16.3 ± 0.7 –8.2 ± 0.9 27 ± 3
TES 0.5 ± 0.1 17.5 ± 0.4 –23.9 ± 0.6 –17.3 ± 0.8 –6.6 ± 1.0 22 ± 3
TAPSO 0.5 ± 0.1 17.4 ± 0.5 –23.7 ± 0.7 –18.1 ± 1.1 –5.6 ± 1.3 19 ± 5
Average 17.6 ± 0.2 –24.0 ± 0.3 –17 ± 1 –7 ± 1 23 ± 2
Enthalpically very favorable• Coordination site is tuned for Cu+Entropically favorable• Cu+ desolvation; displaced protons, but His35 & His83 protonated
Metallothionein ��
MDPNCSCAADGACTCATSCKCKECKCTSCKKSCCSCCPSGCAKCAQGCICKGASDKCSCCA
Relative metal ion affinity:Ni2+~Co2+<Zn2+<Cd2+~Pb2+<Ag+~Cu+<Hg2+
Krezel and Maret have reported that 4 Zn2+ bind with K = 6 x 1011, 2 Zn2+ bind with K ~ 1010 and 1 Zn2+ binds with K = 5 x 107.
Metallothionein-3 ��
MDPNCSCAADGACTCATSCKCKECKCTSCKKSCCSCCPSGCAKCAQGCICKGASDKCSCCA ��
MDPETCPCPSGGSCTCADSCKCEGCKCTSCKKSCCSCCPAECEKCAKDCVCKGGEAAEAEAEKCSCCQ
Collaboration with Rachel Austen (Bates à Barnard) MT-3 is the metallothionein isoform found in neuronal tissue Unique Pro residues in N-terminal β domain and insert in C-terminal α domainHigh affinity for Pb2+ suggests potential role in lead neurotoxicityQuantify Pb2+ binding to MT-3 and competition with Zn2+
Metallothionein-3Pb2+ à Zn7MT3
pH 6.0 , 100 mM MES, 25 ± 0.2 °C, 307 rpm
Best Fit values:nITC = 5.8 ± 0.03KITC = 3.1 (± 0.2) x 105
ΔHITC = -7.16 ± 0.05 kcal/molAverage values:nITC = 6.9 ± 1.5KITC = 3 x 105
ΔHITC = -9 kcal/mol
Metallothionein-3EDTA à Pb7MT3
pH 6.0 , 100 mM MES, 25 ± 0.2 °C, 307 rpm
One set of sites:nITC = 0.50 ± 0.01KITC = 8.3 (± 0.1) x 105
ΔHITC = -7.8 ± 0.1 kcal/mol
Two sets of sites:K1ITC = 3 (± 1) x 108
ΔH1TC = -0.7 ± 0.1 kcal/molK2TC = 1.7 (± 0.2) x 106
ΔH2ITC = -5.0 ± 0.03 kcal/mol
Competition:KEDTA = 1.3 x 1014 (fixed)ΔHEDTA = -11 kcal/mol (fixed)KMT = 8 (± 2) x 1011
ΔHMT = -6.6 kcal/mol (fixed)
Metallothionein-3EDTA à Zn7MT3
pH 6.0 , 100 mM MES, 25 ± 0.2 °C, 307 rpm
One set of sites:nITC = 0.17 ± 0.01KITC = 2.3 (± 0.6) x 106
ΔHITC = -6.5 ± 0.2 kcal/mol
Two sets of sites:K1ITC = 1.3 (± 0.3) x 108
ΔH1TC = +5.14 ± 0.03 kcal/molK2TC = 3.9 (± 0.5) x 105
ΔH2ITC = -4.3 ± 0.1 kcal/mol
Competition:KEDTA = 4.1 x 1012 (fixed)ΔHEDTA = -7.6 kcal/mol (fixed)KMT = 7.8 (± 0.7) x 1010
ΔHMT = -3.2 kcal/mol (fixed)
Metallothionein-3Experimental enthalpies of metal ions binding to MT-3 at pH 6.0,
and analysis to obtain the buffer-dependent binding enthalpies; units of kcal/mol.
Analysis to determine buffer-independent formation enthalpies for M7MT-3 and the formation enthalpies with deprotonated Cys thiols; units of kcal/mol
Metallothionein-3Thermodynamic values for the three populations of metal ions binding to MT-3
at pH 6.0 and 298 K.
a) units of kcal/mol, b) units of cal/mol K
M. C. Carpenter, A. Shami Shah, S. DeSilva, A. Gleaton, A. Su, B. Goundie, M. L. Croteau, M. J. Stevenson, D. E. Wilcox, R. N. Austin, “Thermodynamics of Pb(II) and Zn(II) Binding to MT-3, a Neurologically Important Metallothionein”, Metallomics, 2016, in press
Ln3+-binding peptides
Lanthanide-binding peptides (Lamp) have been selected with phage displayBind to Ln3+-hydroxo species and form insoluble aggregatesPotential applications in separation of Ln3+ ions from seawater and wastewaterAggregates and their formation have been characterized by several methods:
ICP-OES, FT-IR, SEM, TEM, EDX, EXAFS, NMR and ITC
29
624
625
626
Figure 4. Mechanism of Ln3+ mineralization. Schematic illustration of the Lamp-induced 627
mineralization mechanism. i) Interaction of Lamp with [Ln(H2O)8–9]3+
. ii) The high 628
electrophilicity of Ln3+ and nucleophilic attack by Lamp causes deprotonation of coordinated 629
H2O, resulting in generation of Ln hydroxide. iii) Lamp stabilizes Ln hydroxide and prevents 630
dehydroxylation. iv) Hydrophobic effect induces accumulation of Lamp with Ln hydroxide 631
complexes. v) Precipitation of large clusters. 632
633
634
Lamp
(i) Nucleophilic attack
(v) Precipitation(iv) Hydrophobic accumulation(iii) Stabilization of Ln-hydroxide
(ii) Generation of Ln-hydroxide
Ln3+-binding peptidesDy3+ à Lamp-1 Dy3+ à Lamp-2 Nd3+ à Lamp-3
18
Supplementary Figure S17 210
211 Supplementary Figure S17. Thermodynamic analysis of the mineralization reaction. ITC 212 experiments for the reaction of Dy3+ with (a) Lamp-1, (b) Lamp-2, and (c) LBT3, and (d) Nd3+ 213 with Lamp-3. The upper panels show the calorimetric titration profile. The lower panels show a 214 least squares fit of the data to the heat absorbed/mol of titrant versus the ratio of the total 215 concentration of Dy3+ or Nd3+ to the total concentration of peptide. The solid line is the best fit of 216 the data to a single binding site model using a non-linear least squares fit. The thermodynamic 217 parameters are summarized in Supplementary Table S5. 218
0.00
0.05
0.10
0.15
0.20
-0.2
-0.1
0.0
0.1
0.2
0 5 10 15 20 25
0
2
4
0
5
µcal/
sec
a b
kcal/
mol
e of i
njec
tant
kcal/
mol
e of i
njec
tant
0 5 10 15 20 25
µcal/
sec
d
-2 0 2 4 6 8 10 12 14 16
0.0
0.2
0.4
0.6
0.8
0.0
0.5
1.0
1.5
2
Molar Ratio
kcal/
mol
e of i
njec
tant
µcal/
sec
c
0.0
0.5
1.0
1.5
2.0
0.0
0.1
0.2
0.3
0.4
Molar Ratio
kcal/
mol
e of i
njec
tant
0 0.5 1.0 1.5 2.0 2.5
µcal/
sec
18
Supplementary Figure S17 210
211 Supplementary Figure S17. Thermodynamic analysis of the mineralization reaction. ITC 212 experiments for the reaction of Dy3+ with (a) Lamp-1, (b) Lamp-2, and (c) LBT3, and (d) Nd3+ 213 with Lamp-3. The upper panels show the calorimetric titration profile. The lower panels show a 214 least squares fit of the data to the heat absorbed/mol of titrant versus the ratio of the total 215 concentration of Dy3+ or Nd3+ to the total concentration of peptide. The solid line is the best fit of 216 the data to a single binding site model using a non-linear least squares fit. The thermodynamic 217 parameters are summarized in Supplementary Table S5. 218
0.00
0.05
0.10
0.15
0.20
-0.2
-0.1
0.0
0.1
0.2
0 5 10 15 20 25
0
2
4
0
5
µcal/
sec
a b
kcal/
mol
e of i
njec
tant
kcal/
mol
e of i
njec
tant
0 5 10 15 20 25
µcal/
sec
d
-2 0 2 4 6 8 10 12 14 16
0.0
0.2
0.4
0.6
0.8
0.0
0.5
1.0
1.5
2
Molar Ratio
kcal/
mol
e of i
njec
tant
µcal/
sec
c
0.0
0.5
1.0
1.5
2.0
0.0
0.1
0.2
0.3
0.4
Molar Ratio
kcal/
mol
e of i
njec
tant
0 0.5 1.0 1.5 2.0 2.5
µcal/
sec
18
Supplementary Figure S17 210
211 Supplementary Figure S17. Thermodynamic analysis of the mineralization reaction. ITC 212 experiments for the reaction of Dy3+ with (a) Lamp-1, (b) Lamp-2, and (c) LBT3, and (d) Nd3+ 213 with Lamp-3. The upper panels show the calorimetric titration profile. The lower panels show a 214 least squares fit of the data to the heat absorbed/mol of titrant versus the ratio of the total 215 concentration of Dy3+ or Nd3+ to the total concentration of peptide. The solid line is the best fit of 216 the data to a single binding site model using a non-linear least squares fit. The thermodynamic 217 parameters are summarized in Supplementary Table S5. 218
0.00
0.05
0.10
0.15
0.20
-0.2
-0.1
0.0
0.1
0.2
0 5 10 15 20 25
0
2
4
0
5
µcal/
sec
a b
kcal/
mol
e of i
njec
tant
kcal/
mol
e of i
njec
tant
0 5 10 15 20 25
µcal/
sec
d
-2 0 2 4 6 8 10 12 14 16
0.0
0.2
0.4
0.6
0.8
0.0
0.5
1.0
1.5
2
Molar Ratio
kcal/
mol
e of i
njec
tant
µcal/
sec
c
0.0
0.5
1.0
1.5
2.0
0.0
0.1
0.2
0.3
0.4
Molar Ratio
kcal/
mol
e of i
njec
tant
0 0.5 1.0 1.5 2.0 2.5
µcal/
sec
18
Supplementary Figure S17 210
211 Supplementary Figure S17. Thermodynamic analysis of the mineralization reaction. ITC 212 experiments for the reaction of Dy3+ with (a) Lamp-1, (b) Lamp-2, and (c) LBT3, and (d) Nd3+ 213 with Lamp-3. The upper panels show the calorimetric titration profile. The lower panels show a 214 least squares fit of the data to the heat absorbed/mol of titrant versus the ratio of the total 215 concentration of Dy3+ or Nd3+ to the total concentration of peptide. The solid line is the best fit of 216 the data to a single binding site model using a non-linear least squares fit. The thermodynamic 217 parameters are summarized in Supplementary Table S5. 218
0.00
0.05
0.10
0.15
0.20
-0.2
-0.1
0.0
0.1
0.2
0 5 10 15 20 25
0
2
4
0
5
µcal/
sec
a b
kcal/
mol
e of i
njec
tant
kcal/
mol
e of i
njec
tant
0 5 10 15 20 25
µcal/
sec
d
-2 0 2 4 6 8 10 12 14 16
0.0
0.2
0.4
0.6
0.8
0.0
0.5
1.0
1.5
2
Molar Ratio
kcal/
mol
e of i
njec
tant
µcal/
sec
c
0.0
0.5
1.0
1.5
2.0
0.0
0.1
0.2
0.3
0.4
Molar Ratio
kcal/
mol
e of i
njec
tant
0 0.5 1.0 1.5 2.0 2.5
µcal/
sec
Dy3+ à LBT3 (control)
Ln3+-binding peptides
Thermodynamic values from ITC measurements of Ln3+ binding to peptides.
25
293 Supplementary Table S4. Binding strength of synthetic peptides with hydroxylated Ln2O3 294
295 N-terminally biotinylated peptides were used for detection. 296 The EC50 values were obtained from triplicate measurements. 297
298 299 300 301 302 303 304
305 Supplementary Table S5. Thermodynamic parameters of the peptide and Ln3+ reaction 306
307 ǻG was calculated using the equation ǻG = -RT ln K. 308 -TǻS was calculated using the equation ǻG = ǻH - TǻS. 309 R: gas constant. 310 T: absolute temperature. 311 N: reaction stoichiometry. 312 aN is assumed to 1. 313 n.d.: not detected (below the detection limit). 314 315 316 317 318 319 320
0.1 ± 0.01.2 ± 0.3
0.6 ± 0.217.5 ± 6.424.2 ± 12.0 65.5 ± 18.5RE-1
Hydroxylated Dy2O3 Hydroxylated Nd2O3
-
EC50 (µM)
Lamp-1Lamp-2Lamp-3LBT3
--
-
Peptide Target ຒH (kcal/mol) -Tຒ S (kcal/mol) ຒG (kcal/mol) K (x103M-1) N
Lamp-1 Dy3+ 7.34 ± 0.05 -12.79 ± 0.06 -5.73 ± 0.01 15.9 ± 0.35 1a
Lamp-2 Dy3+ 1.35 ± 0.04 -5.63 ± 0.06 -4.42 ± 0.06 1.74 ± 0.10 1a
LBT3 Dy3+ 1.81 ± 0.04 -10.91 ± 0.27 -9.11 ± 0.27 477 ± 174 1.08 ± 0.01
RE-1 Dy3+ n.d n.d n.d n.d n.d
Lamp-3 Nd3+ 2.41 ± 0.09 -7.48 ± 0.11 -5.08 ± 0.06 5.19 ± 0.49 1a
a) assumed to be 1
ITC of Metal-Protein Interactions1. General- know the metal chemistry (e.g., disproportionation of Cu+; solubility)
2. Azurin- delivery of metal in well-defined complex with buffer or chelate
avoids unwanted (unknown) reactions, and allows high affinity binding sites to be studied
- number of protons displaced upon metal binding at a given pH can be quantified with ITC data in different buffers
3. Metallothionein-3- it may be necessary or useful to measure the extraction (chelation) of
the metal from the protein by a chelating agent and analyze with microscopic reversibility, after accounting for chelate-metal thermodynamics (ΔH, K).
4. Lanthanide-binding peptides- correlation between ITC data and molecular processes can be
challenging in complex systems (N. E. Grossoehme, A. M. Spuches, D. E. Wilcox J. Biol. Inorg. Chem. 2010 15, 1183-1191)
Acknowledgements
Yi Zhang G'01Shreeram Akilesh '00
Dr. Anne RichAustin Schenk '00
Nick Grossoehme G’07 (Winthrop U)Dr. Anne Spuches (E Carolina U)
Colette Quinn G’09 (TA Instruments)Jolene Schuster G’09
NIH ES07373
Dartmouth Superfund Research Program
Jessica Son ’11Becky Rapf ’12
Molly Carpenter G’14George Lisi G’14
Max Png ‘14Molly Croteau G’15
*Michael Stevenson G’16*Michael Cukan G’18
NSF CHE 0910746 and CHE 1308598
“Thermodynamics of Metal-Protein Interactions