Chemical Modification

• Variety– Oxidation– Nitrosylation– Dissociation

• Effects– Folding

• Controls– Environmental– 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

• Complexchemistry alterssensitivity

Multiple modifications

• For fixed “A” the amount of product is• One “B”: AB

–

• Two “B”: AB+AB2

–

• More

– Hill equation: • n: cooperativity• Kd: apparent dissociation constant

][1

][

BK

BK

A+AB

AB

2

2

]['1]['

)1(][1

][BKBK

BKBK

nn

nn

n BK

BKa

][1

][

nd

n

xKnx

Chemical sensors

Increasing cooperativity increases gain

Decreasing Kd increasing affinity, increases sensitivity

2-3 log dynamic range

pH

• Charged amino acid side chains

• H+ movement can dramatically alter molecular folding

• pK=-log( )[B-][H+]

[HB]

Bicarbonate buffers

• CO2 solubility ~0.03mM/Torr

• CO2 Hydration

– Carbonic anhydrase–

• Henderson-Hasselbalch equationK=

pK= -log( )

pK = pH – log( )

pH = pK + log( ); pK=6.1

[HCO3-][H+]

[CO2][HCO3-][H+]

[CO2][HCO3-]

[CO2][HCO3-]

[CO2]

Bicarbonate buffers

5% CO2

pH 7.4

pH 7.2pH 2

pH 12 pH 7.6

[HCO3-][H+]

[CO2]Ka =

Open vs Closed Buffer Systems

• Bicarbonate

• Physiological– pCO2 = 40 mmHg– [CO2] = 1.2 mM– [CO2]+[HCO3]=31 mM

• HEPES

• Equivalent Buffer– [HEPES]+[HEPES-]=31 mM– [HEPES-]=14.7– [HEPES]=16.3

][

][log

2

3

CO

HCOpKpH

][

][log

HEPES

HEPESpKpH

][

][log1.65.7

2

3

CO

HCO

][

][log55.75.7

HEPES

HEPES

25][

][

2

3

CO

HCO 9.0][

][

HEPES

HEPES

Open vs Closed Buffers

• Bicarb– Add 10 mM HCl

• Immediate– [HCO3]=20 mM– [CO2]=11 mM– pH = 6.4 (400 nM)

• HEPES– Add 10 mM HCl

• Immediate– [HEPES-]=4.7 mM– [HEPES]=26 mM– pH=7.2 (63 nM)

• Much better pH control near pKa

][

]][[log

2

3

CO

HCOHpK

][

]][[log

HEPES

HEPESHpK

)0012.0(

)030.0)(01.0(log1.6

d

dd

)0163.0(

)0147.0)(01.0(log55.7

d

dd

Open vs Closed Buffer System

• Bicarb

• CO2 solubility 1.2 mM

– [HCO3]=20 mM-350 nM– [CO2] = 1.2mM+350 nM– pH=7.3 (50 nM)

• Much better than 6.4 w/o exchange

• HEPES

• No mass exchange– pH =7.2

][

]][[log

2

3

CO

HCOHpK

]011.0[

]02.0][74[log

epK

]026.0[

]005.0][86[log

epK

)0012.0(

)020.0)(104(log1.6

2

227

d

dd

][

]][[log

HEPES

HEPESHpK

)0012.0(

)030.0)(01.0(log1.6

dd

Could do bicarb in one step:

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

][

]][[log

acid

HbasepK

pKpHacid

base

][

][log

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.

pH control demo

• Talin-dependent adhesion/motility

• Glycolysis

pH dependence of several glycolysis enzymes(Xie et al 2014)

Talin structure

Block NHELower intracellular pH

stabilize FAs

Srivastava et al 2008

Reactive modification

• NO S-nitrosylation– 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

-S-H -S-N=O

Reactive oxidation

• Partly 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

Kung & Bolton 1997

Thymine glycol distorts DNA structure

Thymine glycolThymine

•Amino acid modification changes local polarity•Crosslinking•Strand break

Electrochemistry

• Redox reactions describe electron transfer– Zn + CuSO4Cu + ZnSO4

– Zn + Cu2+Cu + Zn2+

– Zn Zn2+ + 2e- and Cu2+ + 2e- Cu

– 2 GSH + H2O2 GSSG + 2H2O

– 2 GSH GSSG + 2e- + 2H+ and

H2O2 + 2e- + 2H+ 2H2O

Bio

logi

cal

Inor

gani

c

Electrochemistry-free energy

• Electrical– G = -nFE– Faraday constant 9.65 104 C/mol

• Concentration– G = RT ln( Q)– Gas constant 8.31 J/K/mol

• Whole reaction– G = G0 + RT ln(QProd) – RT ln(Qreac)– -nFE = -nFE0 + RT ln(Qprod/Qreac)– E = E0 - RT/nF ln(Qprod/Qreac)

• Nernst Equation for redox reaction• Equilibrium at G= E = 0

Electrochemistry-half cells

• Standard Reduction Potential E0

• Metals (Daniell cell)– Zn Zn2+ + 2e-

Zn2+ + 2e- Zn E0=-0.76V– Cu2+ + 2e- Cu E0=+0.34V– E0=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

– E0=1.78-(0.18) = 1.6V

Cellular Redox State• Biological

– 2 GSH + H2O2 GSSG + 2H2O

– E0= 1.6V– G = -nF (1.6V) + RT ln( )– E = 1.6V – RT/nF ln( )

• Steady state trend– 0 = 1.6 –(8.31*310)/(2*9.6e4) ln( )– = 1052

• ie: Not a lot of free peroxide in a cell• Still needs a catalyst

• Real cells have many potential half-cells

GSSGGSH2 H2O2GSSG

GSH2 H2O2

GSSGGSH2 H2O2GSSG

GSH2 H2O2

GSH:GSSG redox buffer

• GSH is abundant reducing agent• GSSG + 2e- + 2H+ 2 GSH E0=+0.18

– E = 0.18 – RT/nF ln( ) – E = 0.18 – 0.03 log( )

• GSH:GSSG ratio as marker of redox state– More GSH, more negative E, more reducing

• GSSG reduction appears as negative in whole reaction

• Whole reaction more favorable with positive E

– More H+, more positive E, more oxidizing• Neutral [H+]2 ~ 10-14

• Many biological oxidations include H+

GSSG H2GSH2

GSSG H2GSH2

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– H2O2H2O 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

Extracellular antioxidants

Cytoplasmic antioxidantsCytoplasmic oxidants