Bio-Inorganic Chemistry Part 2.ppt · 8/28/2019 1 DR. R. P. JOHN Bio-Inorganic Chemistry...

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D R . R . P . J O H N

Bio-Inorganic Chemistry

Bio-inorganic Chemistry -by R. P. John

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Syllabus

Metal Storage Transport and Biomineralization: Ferritin, transferrin, and siderophores

Calcium in Biology: Calcium in living cells, transport and regulation, molecular aspects of intramolecular processes, extracellular binding proteins

Metalloenzymes: Zinc enzymes-carboxypeptidase and carbonic anhydrase. Iron enzymes-

catalase, peroxidase and cytochrome P-450. Copper enzymes – superoxide dismutase. Molybdenum oxatransferase enzymes- xanthine oxidase. Coenzyme vitamin B12.

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R O L E O F C A L C I U M I N B I O L O G I C A L S Y S T E M S

C A L C I U M T R A N S P O R T A N D R E G U L A T I O N

I N T R A C E L L U L A R P R O C E S S E S

E X T R A C E L L U L A R C A L C I U M B I N D I N G P R O T E I N S

Bio-inorganic Chemistry: PART 2

Learning Objectives

StudentswilllearnroleofCa2+incellularfunction Understanduptakemechanism UnderstandthesignallingmechanismbyCa2+

UnderstandtheenergeticsofCa‐ATPase,CNXetc. KnowvariousCa2+ receptorproteins Understandthestructureandactionofreceptorproteins

ExplainwhyCa2+ isusedformediatingcellularprocess KnowvarioustechniquesusedforestimatingCa2+


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Methods for estimating Ca2+ in Cells


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1. Ca2+ selective electrodes: o Micro electrode made of Micropipette. Has reference electrode and an ion selective

electrode in a double barrel arrangement. The membrane is made of PVC gel containing Ca2+ complexing agent ETH1001

Pros: Can measure up to 10‐8 MCons: Cannot measure transient flux


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2. Bioluminescence: o The light emission in Jelly Fish is due to a protein called ‘Aequorin’. o It happens when a prosthetic group in ‘Aequorin’ returns to ground state from a high energy state, due to a reaction promoted by Ca2+.

o This emission is weak when the Ca2+ concentration is below 0.3M. o The light emission is a function of concentration in the range 0.5 to 1.0 M.

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3. Using complexing agents with Ca2+ dependant light absorption or Fluorescence

o Suitable for measurement of concentration in the 1M to 10nM in the presence of 1mM Mg2+ and 100 mM Na+/K+

Ca2+ dependant light absorption or Fluorescence


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4. Using 19F –NMR of complexing agents with Ca2+

The 19F signals of the Ca2+ bound 5‐F BAPTA, appear 6ppm downfield compared to that of unbound 5F‐ BAPTA

The method can differentiate metal ions, as the chemical shift differ for different species.

The binding constant and Free Ca2+ can be calculated using the eqn. Kb= Kon/Koff

Kon = KB Koff

Measurement of Total Ca concentration


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1. EPMA: Electron Probe X‐ray Micro Analysis

2. EELS: Electron Energy Loss Spectroscopy 3. PIXE: Proton Induced X‐ray Emission4. Ion Microscopy

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PIXE image of a hair of Stinging Nettle


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Color Code:The Yellow: >3.4M, Orange: 2.0‐ 3.4M, Red: 1.‐2.0M, dark blue: 1.0‐1.5, Blue: 0.5‐1.0, light Blue: 0.5‐0.1M, White: .0.1M

Calcium ion Transport


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Bio-inorganic Chemistry -by R. P. John 13


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Ca-ATPase

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SR Lumen

Cytoplasm

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Map view of Ca-ATPase


Calcium -ATPase

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Ca2+ influx by Ca2+ ATPase


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Ca-ATPase: Mechanism

http://www.iam.u‐tokyo.ac.jp/StrBiol/resource/res.html; www.pnas.orgcgidoi10.1073pnas.0506222102

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Calsequestrin

Calsequestrin can bind 18-50 Ca2+ ions

It do so by exposing the charged surface of the protein to Ca2+

M.Wt 40kDa

Plasma memebrane: Ca2+/Na+ Exchanger


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CBD2 site

www.pnas.orgcgidoi10.1073pnas.0707417104

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CaBinding Domain 2 in CNX

Calcium transport: Thermodynamics


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Ca2+ messenger system- Pathway

Bio-inorganic Chemistry -by R. P. John 26Inositol Triphosphate and Ca2+ messenger system

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Heart beat and Calcium: VGCC

The Calcium release associate with Troponin C and cause systole (contraction), release of Calcium from sarcomere cause diastole (expansion)LTCC: L-Type Calcium channels, RyR: Ryanodyne Receptor, -AR: -adrenergic receptor, AC: Adenylyl cyclase, FKBP; Proteins that bind immunosuppressive drugs, function as protein folding chaperons for proteins having prolin residues


Ryanodine

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Neurotransmitter release & Ca2+

Synaptosome Binds to PM via SNAREs. SNARE is made of Synaptobrevin, syntaxin & SNAPs. Binding of Ca2+ by Synaptogamin, coupled with ATP, trigger fusion of synaptosome.

Voltage Gated Calcium Channel –L Type


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Calmodulin: CaM

CaM mediates processes such as inflammation, metabolism, apoptosis, intracellular movement, short and long term memory, nerve growth and immune response.

PDB CODE:1CLL


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CaM-Protein binding

www.pnas.orgcgidoi10.1073pnas.0508640103

SK Channel: It is Ca2+

activated K+ channelGAD: It is a gene

Glutamate Decaboxylase that encodes the enzyme Glutamic acid decarboxylase. GAD is responsible for synthesizing GABA (-aminobutyric acid)

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CaM Kinase binding


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EF-Hand topology of Calmodulin

http://employees.csbsju.edu/hjakubowski/Jmol/Calmodulin_EF_Hand/Calmodulin_EF_Hand.htm

Helix‐loop‐Helix

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Conformation change upon Ca2+ binding

Muscle contraction: Troponin Complex

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Turkey skeletal muscle Troponin C


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Parvalbumin & Calbindin D9k

Parvalbumin: Has 2 main Ca2+ binding sitesRole: buffering Ca2+ in muscle cells

Calbindin 9k: Has 2 Ca2+ binding sitesRole: Acts as Ca2+ carrier/buffer

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Protein Kinase C: Function

• C1 binds phorbol ester• C2 binds phospholopid • C3 & C4 is Kinase domain• Regulation of activity occur through HM turn & V5

Ca2+ selectivity filter


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Phospholipase A2


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Found in mammalian cells, insect and snake venom Hydrolyses 2‐carbonyl ester linkage in glyceric phospho lipids. Release arachidonic acid disproportionately leading to stinging sensation & inflamation

Phospholipase A2: Mechanism

A: Catalytic attack of the substrate bound in productive mode

B: Tetrahedral Intermediate that collapses to give products

C: Products from ‘productive collapse’ where water molecules move in to occupy the active site

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Role of Calcium in Clotting of Blood

‘Pro’ segment of Thrombin


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Platelet activation also induces large morphological changes‐membrane lipids rearrange‐ phosphatidyl serine which is usually on the inner membrane of the platelet, flips out to outer membrane where it plays a role in binding prothrombin.

● Platelet adhesion is mediated by vWF (von Willebrand factor) that binds to both platelet receptors and collagen. This activates plateletscausing release of TxA2 reducing blood flow

● During activation, a receptor for fibrinogen becomes exposed onthe platelet membrane.

● Activated platelets release:

Fibrinogen ADP/ATPvWF SeratoninFactor V Ca2+

Factor VIIIPlatelet derived growth factor (PDGF) ~ promotes healingPlatelet factor IV – prevents formation of active thrombin

inhibitor from heparin and anti‐thrombin III.

dense core granules

‐granules

Mechanism of Blood Clotting

Propeptide of Prothrombin●Propeptide has 3 domains

N‐term 40 residue Gla domain‐ Gla = carboxyglutamate

‐ Strong Ca2+ chelator

‐ Bind Ca 2+ that mediates interaction with phospholipid membrane of platelet C

C

O

CH

O

CH2

CH O-

O-

NH3+

O

O-

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Mechanism of action of Rubisco

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Bio-Inorganic Chemistry Part 2.ppt · 8/28/2019 1 DR. R. P. JOHN Bio-Inorganic Chemistry...

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Transcript of Bio-Inorganic Chemistry Part 2.ppt · 8/28/2019 1 DR. R. P. JOHN Bio-Inorganic Chemistry...