Download Chemical Modification - Georgia Institute of Technology

Chemical Modification
• Variety
– Oxidation
– Nitrosylation
– Dissociation
• Effects
– Folding
• Controls
– Enviromental
– Reactive
Protein structure - function
• AA sequence
– O-N pairing of backbone
– Ionic/hydrophobic interaction of side chains
• Chemical environment
– Ionic strength
– pH – ie: H+ ions
• Reactive
– Side chain modification
Chemistry
• A + B  AB
– AB increases with either A or B
– Equilibrium constant Ka=[AB]/([A][B])
• With total A constant
– A/AB switch
– A/AB indicator
• Complex
chemistry alters
sensitivity
Multiple modifications
• For fixed “A” the amount of product is
• One “B”: AB
–
AB
K [ B]
=Q=
A+AB
1 + K [ B]
• Two “B”: AB+AB2
–
K [ B]
K '[ B ] 2
Q=l
+ (1 - l )
1 + K [ B]
1 + K '[ B ] 2
• More
K n [ B ]n
Q =  an
1 + K n [ B ]n
xn
– Hill equation: Q =
Kd + xn
• n: cooperativity
• Kd: apparent dissociation constant
Chemical sensors
2-3 log dynamic range
Decreasing Kd increasing
affinity, increases
sensitivity
Increasing cooperativity
increases gain
pH
• Charged amino
acid side chains
• H+ movement
can dramatically
alter molecular
folding
[B-][H+]
• pK=-log( [HB] )
Bicarbonate buffers
• CO2 solubility ~0.03mM/Torr
• CO2 Hydration
– Carbonic anhydrase
–
• Henderson-Hasselbalch equation
[HCO3-][H+]
K=
[CO2]
[HCO3-][H+]
pK= -log(
)
[CO2] [HCO3 ]
pK = pH – log( [CO ] )
2 [HCO ]
pH = pK + log( [CO 3] );
2
pK=6.1
Bicarbonate buffers
-][H+]
Ka = [HCO3
[CO2]
pH 2
pH 7.2
5% CO2
pH 7.4
pH 12
pH 7.6
Open vs Closed Buffer Systems
• Bicarbonate
3
[ HCO ]
pH = pK + log
[CO2 ]
[ HCO3- ]
7.5 = 6.1 + log
[CO2 ]
[ HCO3 ]
= 25
[CO2 ]
• Physiological
– pCO2 = 40 mmHg
– [CO2] = 1.2 mM
– [CO2]+[HCO3]=31 mM
• HEPES
-
[ HEPES ]
pH = pK + log
[ HEPES ]
[ HEPES - ]
7.5 = 7.55 + log
[ HEPES ]
[ HEPES - ]
= 0.9
[ HEPES ]
• Equivalent Buffer
– [HEPES]+[HEPES-]=31 mM
– [HEPES-]=14.7
– [HEPES]=16.3
Open vs Closed Buffers
• Bicarb
• HEPES
– Add 10 mM HCl
+
3
[ H ][ HCO ]
pK = - log
[CO2 ]
(0.01 - d )(0.030 - d )
6.1 = - log
(0.0012 + d )
• Immediate
– [HCO3]=20 mM
– [CO2]=11 mM
– pH = 6.4 (400 nM)
– Add 10 mM HCl
[ H + ][ HEPES - ]
pK = - log
[ HEPES ]
(0.01 - d )(0.0147 - d )
7.55 = - log
(0.0163 + d )
• Immediate
– [HEPES-]=4.7 mM
– [HEPES]=26 mM
– pH=7.2 (63 nM)
• Much better pH control
near pKa
Open vs Closed Buffer System
• Bicarb
+
3
[ H ][ HCO ]
pK = - log
[CO2 ]
[4e - 7][0.02]
pK = - log
[0.011]
• CO2 solubility 1.2 mM
6.1 = - log
-7
(4 10 - d 2 )(0.020 - d 2 )
(0.0012 + d 2 )
– [HCO3]=20 mM-350 nM
– [CO2] = 1.2mM+350 nM
– pH=7.3 (50 nM)
• Much better than 6.4
w/o exchange
• HEPES
[ H + ][ HEPES - ]
pK = - log
[ HEPES ]
[6e - 8][0.005]
pK = - log
[0.026]
• No mass exchange
– pH =7.2
Could do bicarb in one step:
6.1 = - log
(0.01 - d )(0.030 - d )
(0.0012)
pH Control
• Cell membranes impermeable to H+
• Compartmentalization of pH
– Cytoplasm 7.15
– Nucleus 7.2
– Mitochondria 8.0
– Golgi ~6.3
– Lysosome 5.5
• Transporters
– H+/K+
– HCO3-/Cl-
Dissociation of amino acid side
chains
 [base][ H + ] 

pK = - log 
 [acid ] 
 [base] 
log 
 = pH - pK
 [acid ] 
In cytoplasm, pH=7.15, so
80% of histidine is in base
form (uncharged).
0.1% of lysine is in its base
form.
In Golgi, pH=6.3, and 40%
of histidine is in base form.
Reactive modification
• NO S-nitrosylation
-S-H
-S-N=O
– Cysteines in hydrophobic acid/base
pockets
– Hemoglobin
• S-NO forms in oxidative environment
• Allows NO release in low oxygen
• Targets vasodilating NO to oxygen starved
tissue
Reactive oxidation
• Part reduced oxygen: O2·, H2O2, OH·
• Protein modification
– Cys, His, Phe, Tyr, Met
• Sulfur
• Ring structures
– Chain break
– Cross-linking
– Chain reaction
• DNA/Lipid modification
Reactive modification
Thymine
Thymine glycol
•Amino acid modification
changes local polarity
•Crosslinking
•Strand break
Thymine glycol distorts DNA
structure
Kung & Bolton 1997
Electrochemistry
Inorganic
– Zn + CuSO4Cu + ZnSO4
– Zn + Cu2+Cu + Zn2+
– Zn Zn2+ + 2e- and Cu2+ + 2e- Cu
Biological
• Redox reactions describe electron
transfer
– 2 GSH + H2O2 GSSG + 2H2O
– 2 GSH GSSG + 2e- + 2H+ and
H2O2 + 2e- + 2H+ 2H2O
Electrochemistry-free energy
• Electrical
– DG = -nFDE
– Faraday constant 9.65 104 C/mol
• Concentration
– DG = RT ln( Q)
– Gas constant 8.31 J/K/mol
• Whole reaction
– DG = DG0 + RT ln(QProd) – RT ln(Qreac)
– -nFDE = -nFDE0 + RT ln(Qprod/Qreac)
– DE = DE0 - RT/nF ln(Qprod/Qreac)
• Nernst Equation for redox reaction
• Equilibrium at DG= DE = 0
Electrochemistry-half cells
• Standard Reduction Potential E0
• Metals (Daniell cell)
– Zn Zn2+ + 2eZn2+ + 2e- Zn E0=-0.76V
– Cu2+ + 2e- Cu E0=+0.34V
– DE0=0.34-(-0.76) = 1.1V
• Biological (glutathione)
– 2 GSH GSSG + 2e- + 2H+
GSSG + 2e- + 2H+  2 GSH
E0=+0.18
– H2O2 + 2e- + 2H+ 2H2O
E0=+1.78
– DE0=1.78-(0.18) = 1.6V
Cellular Redox State
• Biological
– 2 GSH + H2O2 GSSG + 2H2O
– DE0= 1.6V
– DG = -nF (1.6V) + RT ln( GSSG
)
GSH2 H2O2
– DE = 1.6V – RT/nF ln( GSSG
)
2
• Steady state trend
GSH H2O2
GSSG
– 0 = 1.6 –(8.31*310)/(2*9.6e4) ln( GSH
)
2H O
2 2
GSSG
52
– GSH2 H O = 10
2
2
• ie: Not a lot of free peroxide in a cell
• Still needs a catalyst
• Real cells have many potential half-cells
GSH:GSSG redox buffer
• GSH is abundant reducing agent
• GSSG + 2e- + 2H+  2 GSH
E0=+0.18
GSH2
– DE = 0.18 – RT/nF ln( GSSG H2 )
2
GSH
– DE = 0.18 – 0.03 log(
)
2
GSSG H
• GSH:GSSG ratio as marker of redox state
– More GSH, more negative DE, more reducing
• GSSG reduction appears as negative in whole
reaction
• Whole reaction more favorable with positive DE
– More H+, more positive DE, more oxidizing
• Neutral [H+]2 ~ 10-14
• Many biological oxidations include H+
Cellular redox cascade
• Oxygen radicals are not equivalent
• ROS generation
– Mitochondria
– Photons (UV & ionizing radiation)
– Inflammatory cells (NADPH oxidase)
• Radical scavengers
– O2•-H2O2 superoxide dismutase
– H2O2H2O Catalase
– H2O2 + GSH GSSG glutathione peroxidase
– OH• hydroxyl (uncharged OH-)
Redox state
• Intracellular reductive
– Low free oxygen, relatively negative
• Extracellular oxidative
– High O2, relatively positive
Extracellular signals that
promote oxidative stress
Cytoplasmic oxidants
Extracellular antioxidants
Cytoplasmic antioxidants