Coulomb interactions between internal ionizable groups and surface charges in proteins Victor...
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Transcript of Coulomb interactions between internal ionizable groups and surface charges in proteins Victor...
Coulomb interactions between internal ionizable groups and surface charges in proteins
Victor KhangulovAugust 31, 2011
Institute in Multiscale Modeling of Biological interactionsJohns Hopkins University
Laboratory of Dr. Garcia-Moreno
Internal ionizable groups in proteins govern many biochemical processes
• Photoactivation
• Ion homeostasis
• H+ transport
• e- transfer
• Catalysis
Most ionizable residues are located on the protein surface.
Internal ionizable residues are responsible for energy transduction:
Can we engineer useful enzymes?
Structure-based Calculations:
• Overestimate electrostatic energies• Underestimate protein polarizability
+-
Background
• pKa values
• Dielectric constants
• Factors that determine pKa values of internal ionizable groups
• Measurement of pKa values of internal ionizable groups
• pKa values in SNase?
+
pKa = 10.4
pKa = 8.0
Shift in a pKa value is related to the Gibbs free energy (ΔG°)
ΔG° total
= 1.36 (10.4 – 8.0)
= 3.2 (kcal/mol)
Self and Coulomb Energies
+-
+𝐺𝑖 ,𝑖° (𝑘𝑐𝑎𝑙 /𝑚𝑜𝑙)=332𝑍2
2𝑟 𝑖𝑜𝑛 𝜀𝐺𝑖 , 𝑗° (𝑘𝑐𝑎𝑙 /𝑚𝑜𝑙)=
332𝑍 𝑖𝑍 𝑗
𝑟 𝑖 , 𝑗𝜀
Coulomb Energy Self Energy
ε εri,j
++εinεout
∆𝐺𝑖 , 𝑖°
+-
+εout εin
∆𝐺𝑖 , 𝑗°
What is a dielectric constant (ε)?
A macroscopic parameter that describes the polarizability of a material
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E
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Parallel plate capacitor
𝜀=𝐶
𝐶𝑉𝑎𝑐𝑢𝑢𝑚ε
0
80
40
20
10
30
50
60
70
Water
Vacuum (ε = 1)
Dry protein powder
Solid amides
DMSO
Dielectric Constants of Materials (2011). Clipper Controls
Liquid amides
Dependence of Coulomb energy on the dielectric constant (εp)
+-
ri,j = 3 Å
ri,j
ri,j = 12 Å
ε
pKa values of internal ionizable groups: Ideal Case
Background
L25K∆ ∆𝐺𝑝𝐻=∆∆𝐺𝑐−𝑅𝑇𝑙𝑛 [ 1+𝑒𝑧 ∙ 2.303 ∙ (𝑝𝐾𝑎
𝐷−𝑝𝐻 )
1+𝑒 𝑧 ∙ 2.303∙ (𝑝𝐾 𝑎𝑁 −𝑝𝐻 ) ]
Titration of Lys-25 in L25K variant in staphylococcal nuclease
Isom DG (2011). PNAS
A systematic study of pKa values of internal ionizable residues in staphylococcal nuclease
Isom DG (2011). PNAS
pKa values of internal groups are anomalous
Isom DG (2011). PNAS
Asp, Glu, and Lys at 25 internal positions in SNase
Measurement of pKa values: Ideal Case
Background
L25K • Assumption: Group behaves independently
• In “ideal” case, internal Lys does not affect pKa values of other ionizable groups
Isom DG (2011). PNAS
Measurement of pKa values: Non-Ideal Case
Isom DG (2011). PNAS13
Background
L25K
Background
T62K
Lys-25Lys-62
Goals of this study
• Determine the extent to which surface charges can be sensed inside a protein
• Examine the effect of surface charges on pKa values of internal ionizable groups
• Determine dielectric properties that govern communication between internal and surface charges in proteins
Variants of SNase where internal Lys is coupled to other ionizable groups
Isom DG (2011). PNAS
Factors that can affect pKa values in proteins
Coupling between internal and surface ionizable residues
Background
T62K
Shift in the pKa of one Lys only(Lys pKa
D = 10.4)
pKaN = 8
Coupling between internal and surface ionizable residues
Background
T62K
Shift in the pKa of Lys and His(Lys pKa
D = 10.4, His pKaD = 6)
His pKa,2N shifted down
Interactions between Lys-62 and surface residues
• What is the microenvironment of Lys-62?
• Which groups are interacting with Lys-62?
• What is the magnitude of these interactions?
• What are the structural consequences of ionization of Lys-62?
• Can these interactions be reproduced self-consistently with continuum electrostatics calculations?
Lys-62 is buried , 6.3 Å from Asp-21 and 8.4 Å from Asp-19
T62K (PHS)
PDB: 3DMU
Resolution: 1.80Å
R-work: 0.198
R-free: 0.255
D21N Substitution Affects pKa of Lys-62
Reference
T62K
Lys-62 pKa = 8.1 0.1
Isom DG (2011). PNAS
His-8
His-121
D21N Substitution Affects pKa of Lys-62
Reference D21N
T62KD21N/T62K
Lys-62 pKa = 8.1 0.1 Lys-62 pKa = 6.8 0.1
Isom DG (2011). PNAS
pKa = 8.10 ± 0.01(pKa = 8.1 ± 0.1 by linkage analysis)
T62K
Depressed pKa of Lys-62 confirmed by NMR spectroscopy
∆(1 H
N)
Che
mic
al S
hift
(pp
m)
24
pKa = 6.71 ± 0.03(pKa = 6.8 ± 0.1 by linkage analysis)
D21N/T62K
pH
∆(1 H
N)
Che
mic
al S
hift
(pp
m)
pKa of Lys-62 is depressed further in the D21N variant of SNase
Lys-62 reports on strong interaction with Asp-21 (1.9 kcal/mol)
+ +
+
- -pKa2 = 8.1
pKa1 = 6.7
∆G = 1.36 (pKa2 – pKa
1)
= 1.36 (8.1 – 6.7 )
= 1.9 kcal/mol
Coupling between Lys-62 and Asp-21
CBCGCO experiments probe the side chain carboxylic groups directly
Titration of Asp-21 shows dependence on the presence of Lys-62
Asp-21 in reference proteinpKa = 6.56 ± 0.02n = 2.00 ± 0.02
Asp-21 in T62KpKa = 4.17 ± 0.08n = 1.14 ± 0.13
Castañeda CA (2009). Proteins
Asp-21 reports on strong interaction with Lys-62 (3.3 kcal/mol)
+ +
+
- -
pKa2 = 6.6
pKa1 = 4.1
∆G = 1.36 (pKa2 – pKa
1)
= 1.36 (6.6 – 4.2 )
= 3.3 kcal/mol
Coupling between Lys-62 and Asp-21
Asymmetry in ∆Gij reported by Asp-21 and Lys-62
+ +
+
- -
pKa2 = 6.6
pKa1 = 4.1
∆Gi,j (Lys-62) = 1.9 kcal/mol
∆Gi,j (Asp-21) = 3.3 kcal/mol
Coupling between Lys-62 and Asp-21
pKa2 = 8.1
pKa1 = 6.7
Which ∆Gij correctly reflects coupling between Lys-62 and Asp-21?
Asp-21 Lys-62
HisAsp/Glu Lys
∆Gi,j (Asp-21) = 3.3 kcal/mol
∆Gi,j (Lys-62) = 1.9 kcal/mol
What other pKa values are affected by Lys-62?
T62KHis-8 -0.10 ± 0.02
Glu-10 -0.26 ± 0.13Asp-19 -Asp-21 -2.39 ± 0.01Asp-40 0.44 ± 0.10Glu-43 0.13 ± 0.04Glu-52 0.16 ± 0.08Glu-57 0.10 ± 0.09Glu-67 0.16 ± 0.12Glu-73 0.09 ± 0.02Glu-75 -Asp-77 -Asp-83 -Asp-95 -Glu-101 0.14 ± 0.10His-121 -0.09 ± 0.01Glu-122 0.03 ± 0.09Glu-129 0.14 ± 0.09Glu-135 -0.01 ± 0.08Glu-142 0.05 ± 0.04Asp-143 0.16 ± 0.10Asp-146 0.06 ± 0.05
ΔpKa between reference and variant protein with Lys-62
Is the interaction between Lys-62 and Asp-21 through protein or through solvent?
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-100 mM Salt 1M Salt
• Charge-charge interaction is shielded by high salt.
• pKa of Asp-21 returns to its “normal” value in the reference protein
Asp-21 in T62K pKa = 4.2
Case 1:ThroughProtein interaction
Case 2:ThroughSolvent interaction
• Interaction not shielded by high salt
• pKa of Asp-21 remains low
pKa = 4.2
pKa 4.2
pKa 6.1
Is the interaction between Lys-62 and Asp-21 through protein or through solvent?
pKa of Asp-21 in T62K
100 mM Salt 1M Salt
6.6 6.1pKa of Asp-21 in Reference protein
4.2 3.8
+
-Lys-62 interacts with Asp-21 through protein
34
Native structure is not perturbed by neutral Lys-62 in the crystal structure
Background (PHS)PDB: 1EY8Resolution: 1.75ÅR-work: 0.187R-free: 0.240
T62K (PHS)PDB: 3DMUResolution: 1.80ÅR-work: 0.198R-free: 0.255
Structure (as reported by TALOS+) is not perturbed by charged Lys
∆+PHS at pH 4.66
T62K at pH 4.52
Chemical Shifts:(C,CA,H,HN,HA)
Difference of > 30° in φ or ψBetween background and T62K
T62K residues in intermediateExchange.
Can structure-based calculations reproduce pKa values of Lys-62?
pKa in T62K: 8.1
pKa in D21N/T62K: 6.7
pKa in D19N/T62K: 7.5
Lys-62 experimental pKa values
Dielectric constant of 10.3 to11.5 reproduced pKa of Lys-62.
FDPB (UHBD), FULL/PARSE, Probe =1.4Å, Stern layer = 2.0ÅFitch CA (2002). Biophysical JournalKarp DA (2007). Biophysical Journal
Structure-based continuum calculations cannot reproduce pKa values and interactions
ΔGi,j(Asp-21 to Lys-62) = 1.9 kcal/mol
ΔGi,j(Asp-19 to Lys-62) = 0.8 kcal/mol
Calculated ΔGi,j value between Lys-62 and Asp-19 and Asp-21
εin required to reproduce ΔGi,j
6.7 (Asp-21 with Lys-62)> 9.5 (Asp-19 with Lys-62)
Conclusions
• Most pKa values are unaffected by Lys-62.
• There is a strong interaction between the internal Lys-62 and Asp-21.
• There is a weaker (but still strong) interaction between Lys-62 and Asp-19.
• The interaction between Lys-62 and Asp-21 appears to be through protein
• Therefore, the interaction between Lys-62 and Asp-21 is predominantly Coulomb in nature
• pKa and ΔGij cannot be reproduced with the same dielectric constant
Variants of SNase that where internal Lys titration is linked to other ionizable groups
Isom DG (2011). PNAS
pKa = 7.1 pKa = 7.2
pKa = 7.7
Internal Lys-34, Lys-36, and Lys-104 do not perturb the protein structure
Reference (3BDC)
F34K (3ITP)
L36K (3EJI)
L104K (3C1F)
Lys-34, 36, and 104 are buried in very different environments
F34K (3ITP) L36K (3EJI) L104K (3C1F)
Residues within 3.5 Å of buried Lys
Residues within 6.4 Å of buried Lys
Titration of His was virtually insensitive to the presence of internal charged Lys
His-8 His-121
Reference F34K L36K V104K
Largest shift of 0.3 pKa units was observed forHis-121 in F34K variant of SNase
Lys-34 has a weak interaction with Asp-21
Lys-34 and Asp-21 are 14 Å apart
Lys-34 with Asp-21pKa = 7.1 ± 0.1
Reference
F34K
Lys-34 has a weak interaction with Asp-21
Lys-34 and Asp-21 are 14 Å apart
Lys-34 with Asp-21pKa = 7.1 ± 0.1
Lys-34 w/o Asp-21pKa = 6.5 ± 0.6
0.8 kcal/mol
D21N
D21N/F34K
Titrations of Asp and Glu residues in the F34K variant
Reference F34K D21N/F34K
pKa = 6.60 ± 0.37
• Glu-10, Asp-77, Asp-82 and Glu-122 report on weak interactions with Lys-34
• Titration of Glu-122 may be reporting on the pKa of Lys-34 in D21N/F34K variant
• Since changes are globally distributed, interaction cannot be determined to be exclusively Coulomb in nature
What about other pKa values in F34K?
F34KHis-8 -0.16 ± 0.01
Glu-10 -0.04 ± 0.03Asp-19 0.31 ± 0.03Asp-21 -0.05 ± 0.01Asp-40 0.05 ± 0.10Glu-43 0.03 ± 0.04Glu-52 0.06 ± 0.08Glu-57 0.03 ± 0.09Glu-67 0.04 ± 0.12Glu-73 -0.04 ± 0.02Glu-75 -Asp-77 -Asp-83 -Asp-95 -0.04 ± 0.09Glu-101 0.19 ± 0.10His-121 0.28 ± 0.03Glu-122 -0.07 ± 0.08Glu-129 0.30 ± 0.09Glu-135 0.04 ± 0.08Glu-142 0.07 ± 0.05Asp-143 0.11 ± 0.02Asp-146 0.05 ± 0.01
ΔpKa between reference and variant protein with Lys-34
Ionization of Lys-34 contributes to globally distributed changes in the HSQC
Peaks not assigned
Peak broadening
Changes in Shifts > 1ppm
Chemical shifts were compared 0.5 pH unitsAbove and below pKa of Lys-34 (7.1)
Lys-36 exhibits high complexity in the ΔΔG vs. pH data
Lys-36 and Asp-21 are 6.3 Å apart
Lys-36 in L36KpKa = 7.2 ± 0.1
Reference
L36K
pKa values of Asp, Glu and His are not perturbed in L36K
L36KHis-8 -0.01 ± 0.01
Glu-10 -0.01 ± 0.04Asp-19
Asp-21
Asp-40 -0.05 ± 0.03Glu-43 0.00 ± 0.01Glu-52 -0.03 ± 0.02Glu-57 -0.02 ± 0.01Glu-67 -0.11 ± 0.04Glu-73 -0.16 ± 0.04Glu-75 -0.13 ± 0.12Asp-77 -Asp-83 -Asp-95 -Glu-101 0.09 ± 0.01His-121 0.01 ± 0.03Glu-122 -0.04 ± 0.02Glu-129 0.03 ± 0.01Glu-135 0.00 ± 0.02Glu-142 -0.09 ± 0.01Asp-143 -0.10 ± 0.03Asp-146 0.07 ± 0.03
ΔpKa between reference and variant protein with Lys-36
Lys-36 exhibits high complexity in the ΔΔG vs. pH data
Lys-36 and Asp-21 are 6.3 Å apart
Lys-36 in L36KpKa = 7.2 ± 0.1
Lys-36 in D21N/L36KpKa = 5.5 ± 1.1
D21N
D21N/L36K
2.3 kcal/mol
Ionization of Lys-36 does not perturb the structure of the protein
Peaks not assigned
Peak broadening
Changes in Shifts > 1ppm
Chemical shifts were compared 0.5 pH unitsAbove and below pKa of Lys-36 (7.2)
Lys-104 ΔΔG vs. pH data suggests significant interaction between Lys-62 and Asp-21
Lys-104 and Asp-21 are 9.3 Å apart
Lys-104 in V104KpKa = 7.7 ± 0.1
Reference
V104K
Lys-104 ΔΔG vs. pH data suggests significant interaction between Lys-62 and Asp-21
Lys-104 in V104KpKa = 7.7 ± 0.1
Lys-104 in D21N/V104KpKa = 6.10 ± 0.84
D21N
D21N/V104K
Lys-104 and Asp-21 are 9.3 Å apart
2.2 kcal/mol
Titration of Asp-21 reports no interaction with Lys-104
Reference V104K D21N/E101Q/V104K D21N/V104K/E122Q
• pKa of Asp-21 increased in V104K suggesting no direct interaction
• Titrations of Glu-101, Glu-122 and Glu-129 are consistent with weak interactions with Lys-104.
Only pKa of Asp-21 is perturbed in V104K
V104KHis-8 -0.01 ± 0.01
Glu-10 -Asp-19 -Asp-21 0.28 ± 0.04Asp-40 0.03 ± 0.01Glu-43 0.05 ± 0.01Glu-52 -0.04 ± 0.03Glu-57 0.11 ± 0.01Glu-67 0.09 ± 0.09Glu-73 0.00 ± 0.07Glu-75 -0.13 ± 0.12Asp-77 -Asp-83 -Asp-95 -Glu-101 0.03 ± 0.01His-121 0.01 ± 0.03Glu-122 -0.11 ± 0.02Glu-129 -0.24 ± 0.02Glu-135 0.03 ± 0.01Glu-142 -0.09 ± 0.01Asp-143 0.13 ± 0.01Asp-146 0.09 ± 0.01
ΔpKa between reference and variant protein with Lys-104
Ionization of Lys-104 contributes to local changes on the HSQC
Peaks not assigned
Peak broadening
Changes in Shifts > 1ppm
Chemical shifts were compared 0.5 pH unitsAbove and below pKa of Lys-104 (7.7)
Calculations exaggerate Coulomb interactions between internal and surface groups
F34K L36K V104K
pKa
ε ε ε
Coulomb contribution
Exp. pKa = 7.1
εapp= 8.0
Exp. pKa = 7.2 Exp. pKa = 7.7
εapp= 6.5 εapp= 3.5
FDPB (UHBD), FULL/PARSE, Probe =1.4Å, Stern layer = 2.0ÅFitch CA (2002). Biophysical JournalKarp DA (2007). Biophysical Journal
Calculations using the εapp that reproduced ΔGi,j , overerestimate shifts in pKa values.
Very strong interactions between groups are possible in ri,j < 10 Å
Conclusions
• Most interactions between internal and surface groups are very weak
• Strong interactions are possible in ri,j < 10Å
• pKa of internal groups can be modulated by surface charges
• pKa and ΔGi,j cannot be reproduced self-consistently using the same dielectric constant
ThanksBGME Lab
Dr. Carolyn FitchDr. Ana Damjanović
Aaron RobinsonBrian DoctrowAlfredo CaroPeregrine Bell-Upp
Dr. Daniel IsomDr. Michael HarmsDr. Michael ChimentiDr. Brian CannonDr. Carlos Castañeda
USNADr. Jamie Schlessman
Support Team
Jerry LevinChris AltizerJessica BaileyRanice CrosbyKen Rutledge
Jenkins Faculty
Dr. George RoseDr. Doug BarrickDr. Greg BowmanDr. Richard ConeDr. Juliette LecomteDr. Sarah WoodsonDr. Karen FlemingDr. Ananya Majumdar
Advisor
Dr. Bertrand García-Moreno E.
Analysis of CBCGCO Titration Curves
)(
)(
101
10)(
a
a
pKpHn
pKpHnAAH
obs pH
Castañeda CA et al. (2009) Molecular determinants of the pKa values of Asp and Glu residues in staphylococcal nuclease. Proteins 77:570-88. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19533744.
Two-Site Binding Isotherm:
)2()(
)2()(2
211
211
10101
1010)(
aaa
aaa
pKpKpHpKpH
pKpKpHA
pKpHAHAH
obs pH
Modified Hill Equation: