"Proteins are Charged: Get Over it" or "What's a nice Arg like you bringing your charge into a place

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"Proteins are Charged Get Over it?"(or)"What's
a nice Arg like you bringing your charge into a
place like this?"(or else)"Proteins it's the
protons, stupid".(or maybe)Proteins are a hot
and complexplace if you're a charge
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As has been said previously
Norah "Everything we selected was neutral
because we didn't want to go there." Geof
"Having formal charges puts us out on a
limb" Well for some problems there is just no
choice
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Buried ionizable residues Transmembrane proteins
e-
Bacteriorhodopsin
Oxidase
H
H
Or transport Cl the other way
Song Y., Gunner M.R. 2003. Biochemistry 42
9875-88.
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BM -212 mV
BL -30 mV
HM 163 mV
HL 240 mV
Gunner, M.R., Nicholls, A., and Honig, B. (1996)
J. Phys. Chem., 100, 4277-91.
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Ionized groups in proteins influences Protein
association with protein, nucleic acids,
membranes Binding ligands - substrate, metals,
cofactors Catalysis - transition state
stabilization Function of transmembrane ion
channels and pumps Electron and proton transfer
proteins (Redox chemistry)
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How to calculate ionization Yifan Song Junjun
Mao Distribution of buried ionizable residues in
the PDB databank Jinrang Kim
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How to calculate the ionization in the
protein?(MCCE)Multi Conformation Continuum
Electrostatics
Bob "full electrostatics is slow" - well with
conformations its slower (advantage pitfall of
academia)
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Electrostatic energies pair-wise solvation)
MM energies Lennard Jones torsion
Reference pKs and redox Ems Sample
conformation and ionization states together with
Monte Carlo Get equilibrium properties not
kinetics Assume chemistry, known in some
reference solvent is perturbed by protein
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MCCE degrees of freedom atomic position and
charge
Ionizable sidechains ligands A- and AH B and
BH
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What's in MCCE?
Alexov, E. and Gunner, M.R. (1997) Biophys. J.
74, 2075-2093
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Repacking the side chains reveals only few low
energy packings
Backbone fixed
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The multi-conformation can be further refined
Backbone fixed
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Hydrogen atoms are added to create ionization
conformers
Backbone fixed
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Junjun MaoYifan Song
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This scales a N (at worst N2) Pre-select
conformers Make look-up table of pair-wise and
self-interactions In MC calculate micro-state
energies from look-up tables Most expensive
part Electrostatics (e inside 4) One PB run (to
proper scale)/conformer for pair-wise Second PB
run/conformer for solvation energy (better
correction for surface anomalies of having all
conformers present for each PB run) It's no
longer 2n microstates. Now
microstates
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Lysozyme (2.4Ghz Xion)

• Prep (5s)
• Conformer generation (30 min)
• Generate non-clashing conformers (23,041)
• 1000 repacks (410 conf)
• Add protons (1020 conf)
• Delphi (5.6 hrs) (once you have the lookup tables
  you can do lots of stuff)
• Monte Carlo 14 pH's (25 min)

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Benchmark Side chain Packing
1JSF
All residues
Buried residues
RMSD in Å
pH
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Structure changes with pH
Nuclease, 3rn3
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Benchmarks pKa
Calculated
Experimental pKa
107 pKa values from 7 proteins (1a2p, 1pga, 1ppf,
4pti, 3icb, 3rn3, 2rn2)
Georgescu R.E., Alexov E.G., Gunner M.R. 2002.
Biophys J. 831731-48.
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ab-roll a-globin 4-helix bundle
Mainly b-barrel
Heme Heme
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Sample Application Em of cytochromes
Em calculations on 16 cytochromes.
Biochemistry, 42 (33), 9829 -9840, 2003
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Buried ionizable residues Transmembrane proteins
e-
Bacteriorhodopsin
Oxidase
H
H
Or transport Cl the other way explain how with 3
mutations you can turn a H pump into a Cl pump
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Changes in conformation or ionization on ET
Alexov E., Gunner M. 1999. Biochemistry 388253-70
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QB
QB
Ser
Ser
Asp-
AspH
AspH
Asp-
Two different states stabilized No proton uptake
from solution
E.Alexov
Alexov E., Gunner M. 1999. Biochemistry 388253-70
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Conclusions 1

• With one set of parameters we can calculate
  experimental pK and redox Em's assaying stability
  of charges in proteins
• The packing matches the crystal structure best at
  the pH of the crystal structure (preliminary
  studies).
• The method allows the protein to change side
  chain ligand position as the pH or reaction
  state changes.
• We don't have to use a generalized higher value
  of e (eg e20) for the protein to get good
  agreement with data.
• The effective dielectric will depend on the local
  protein structure.

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Georgescue BJ 2002 pKs
RCs, Biochem 1999 pKs BJ 2002
EMIL ALEXOV
Yifan Song
Junjun Mao
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How to calculate ionization Yifan Song Junjun
Mao Distribution of buried ionizable residues in
the PDB databank Jinrang Kim
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How hard is it to bury ionizable residues? Use
statistics to judge difficulty How likely is
it that they will in fact be ionized? Use
calculation to determine ionization
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Are these ionizable residues buried?
Define buried
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Definition of Burial Loss of Solvation Energy
Move from water to e4 (Fully bury)
17.4 kcal
1.7 kcal
Removing a charge from water shifts pKs and other
measures of the stability of charged form In a
simple medium this stabilizes the neutral form
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How many are buried?
ASP
ARG
467 (13.6)
589 (20.3)
Fraction
GLU
LYS
127 (3.3)
331 (8.5)
6.8 kcal/mol
Desolvation penalty (1pK1.36Kcal/mol)
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HOW DOES FRACTION BURIED COMPARE WITH
DESOLVATION PENALTY?
Asp
Glu
BURIED
Fraction residue surface exposed
Arg
Lys
DelPhi desolvation penalty
1?pK unit 1.36 kcal/mol
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HOW MANY BURIED DEPENDS ON PROTEIN SIZE
lt100 res Av 1.4/100
101-300 res Av 2.6/100
Number of Proteins
S
gt300 res Av 4.6/100
M
L
BURIED IONIZABLES/100 RES
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FOR THE 513 PROTEINS STUDIED
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Compostion of Proteins 318 Proteins 60,127
Residues 14,126 Ionizable residues 1,514
Buried Ionizable residues
Buried LYS (127/38303.3)
Buried ARG (589/296120)
OTHERS (76.5)
LYS (6.37)
ARG (4.92)
GLU (6.49)
ASP (5.71)
Buried GLU (331/39018.5)
Buried ASP (467/343413.6)
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Are the buried ionizables charged or neutral? (at
pH 7)
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Ionization states at pH7
ARG
ASP
GLU
LYS
Fraction Ionized
Deeply Buried
Mostly charged
Desolvation penalty (1pK1.36Kcal/mol)
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gt5?pK unit desolvation penalty gt90 ionized at pH
7 Arg 99.7 Asp 88.8 Glu 80.0 Lys 76.3 All
89.7
Wide range of interactions with backbone and
sidechains Mostly favorable
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How can a deeply buried charge stay ionized?
If you bury a charge it looses interaction with
water. If it stays ionized the protein must
replace the favorable interactions. We will
divide these into (Backbone dipoles) (Side
chains or Bound ligands)
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Protein is not a simple medium
(Non-polar)
?G
Increasing response to charge
Rigid protein
Protein
??G
Backbone Dipoles (av 90 mV)
Buried Charges (30 of all Charges)
Av Protein Response (e4?)
Rotation dipole
Protonation nearby
Protonation cofactor
(Gunner et al B.J. 2000)
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The most prevalent polar group in the protein is
the amide dipole
Should the average potential from the backbone be
pos. neg. or zip?
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4 proteins with different motifs
Backbone always makes the inside positive!
305 proteins with lots of folds
b
a
a/b
ab
Biophys. J.(2000) 78, 1126-1144.
90 mV 2 kcal/mol/e
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DEEPLY BURIED CHARGED RESIDUES
BACKBONE INTERACTION ENERGY DISTRIBUTIONS
Big range Few unfavorable Acids better stabilized
favorable
D
E
K
R
Backbone interaction
1?pK unit 1.36 kcal/mol
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DEEPLY BURIED CHARGED RESIDUE Side chain side
chain INTERACTIONS
Few unfavorable Big range (ion-pair not
required)
favorable
E
K
D
R
SSidechain interaction
1?pK unit 1.36 kcal/mol
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Interactions of buried charge groups with
backbone and other side-chains
ASP
ARG
Backbone Interactions
LYS
GLU
Pairwise Interactions
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D
R
E
K
For buried charged residues The smaller the side
chains interactions the more favorable the
backbone interactions The bigger the side chain
interactions the more the protein shifts the pK
to stabilize the charged form
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Ionization states at pH7
ARG
ASP
GLU
LYS
Fraction Ionized
Deeply Buried
What differentiates the ionized and neutral
residues?
Desolvation penalty (1pK1.36Kcal/mol)
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BURIED CHARGED VS. NEUTRAL RESIDUES
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For the ionized residues vs neutral buried
ionizables The desolvation energy is a bit
smaller The backbone interaction is a bit
bigger The side chain interactions are a lot
bigger
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If it's buried it's likely to be conserved
Desolvation penalty
Desolvation penalty
lt5?pK
gt5?pK
Fraction
0 5 10 15 20 25 ()
0 10 20 30 ()
Weight Conservation (1.0100 conserved)
Buried ionizables are highly conserved (HSSP)
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513 PROTEINS 88,916 RESIDUES 20,804 IONIZABLE
RESIDUES (23.4) 2,690 BURIED IONIZABLES (12.5)
gt5?pK unit desolvation penalty gt90 ionized at pH
7 Arg 99.7 ionized Asp 88.8 Glu 80.0 Lys
76.3 All 89.7
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How hard is it to bury ionizable residues? From
the data bank 13 of ionizable residues are
buried (Av 3/100 res) How likely is it that
they will in fact be ionized? 90
ionized Proteins tend to stabilize ionization
over what's found in water (lower acid pKas
higher base pKas)
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So

• Buried ionizables are not so rare
• Proteins (which are very polar) can stabilize
  their charge
• The MCCE method can calculate the thermodynamic
  properties (Em's pK's) of groups inside proteins.
• We will have a pK data base up (hopefully) this
  summer for perusal disproval.

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Jinrang Kim Buried charges
Yifan Song MCCE
Junjun Mao MCCE
NIH/NSF/USDA
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Heme Electrochemistry Benchmark
Basic reaction
Move reaction into protein
ab-roll a-globin 4-helix bundle
Mainly b-barrel b5 c, c2
b562, c' f
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Hemes in cytochromes
Calculated Em
Experimental Em
Mao Hauser Biochem Aug 2003
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What's important?
Mao J., Hauser K., Gunner M.R. 2003.
Biochemistry 42 829-40.