Entner -Doudoroff Pathway

Entner -Doudoroff Pathway- (By Michael Doudoroff and Nanthan Entner )

It is an alternative pathway for production of puruvate from glucose. It is different from glycolysis pathway. It uses many enzymes from both Pentose phosphate pathway and glycolysis.

It occurs in prokaryotes specially in the Gram (-)ve bacteria for example pseudomonas aeruginosa, Azotobacter,Rhizobium etc. It absent in eukaryotes.

In this pathway glucose-p is oxidised to 2-keto -3-deoxy -6-phosphogluconic acid. It is cleaved by 

 2-keto -3-deoxyglucose -phosphate aldolase to pyruvate and glyceraldehyde -3-phosphate .

It thrn oxidised to pyruvate by glycolytic pathway . 2 ATPs are produced by substrate level phosphorylation.





                                 Image source -http://microbiochem.weebly.com/e

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EtBr

EtBr- It is a planar ,aromatic,intercatalytic agent. It is used in molecular biology labs as a fluroscent dye for nucleic acid gel electrophoresis.

It binds non covalently to ds Dna by intercatalyting into the stacked base pairs. It forms close vanderwall contacts with the base pairs of  DNA and interact with hydrophobic interior of DNA molecule. 

One EtBr molecule integrated upto 2.5 bp of DNA and causes the DNA to unwind by 27°C in covalently circular DNA .Hence, it decreses the number of helical turns. When EtBr binds to linear DNA it causes the helical pitch to increase.

Effects-
1.Ethidium Bromide (EtBr) is sometimes added to running buffer during the 
separation of DNA fragments by agarose gel electrophoresis.The mobility of 
DNA was always less in the gels with EtBr.

2.Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm. It 
can absorb energy from nucleotides excited by absorbance of 260 nm radiation.

3.In covalently-closed circular DNA(plasmid), twist (turns)and writhe(supercoiling) are directly related.  With ethidium, the negatively supercoiled plasmid ( E. coli) becomes positively supercoiled, and each additional ethidium incorporated further positively supercoils it, like winding a telephone wire.

                     Image source-http://sandwalk.blogspot.in/2007/07/ethidium-bromide-binds-to-dna.html
1.Ethidium Bromide is used in electrophoresis of DNA fragments because:
[a] It makes the fragments visible under UV light
[b] It makes the DNA fragments more mobile in the gel
[c] It increases the conductivity of electricity through the gel
[d] It helps determine the size of the fragments
Ans.  A

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Edman degradation

Edman degradation -(Pher Edman)

Definition-It is a method to determine the sequence of peptides  and proteins from the N- terminal of a polypeptide chain.
Reagent-  Phenylisothiocyanate

Mechanism-

 1.Under alkaline condition-

• Phenylisothiocyanite reacts with N-terminal of amino group of polypeptide .
• The product of the above reaction is phenylthiocarbamyl-peptide derivative (PTC-peptide.)

2. Under acidic condition-

• In the presence of trifluroacetic acid this product cleaved as a thiazolinone derivative.
• It then form phenylthiohydantoin which can be easily identified by using chromatography or electrophoresis.

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Molecular chaperones

Molecular chaperones- 

The spontenity folding of protein is not takes place in case of all the proteins. Some proteins need  a class of  other proteins  for a proper folding. The other proteins are called chaperones.

It is present in both prokaryotic and eukaryotic structures. Chaperones were 1st identified in bacteria E.Coli.

1.Hsp 60( 60 kDa) [ GroEL/GroES in E.coli ] Also called chaperonins forms barrel shaped structure.

GroEL (Larger heptameric structure with 14 identical residues )HSP60 in E.coli)binds to many unfolded proteins . Interaction with the substrate is based on hydrophobic interactions between surfaces of the substrate and residues of GroEL that are exposed in its central cavity. ATP and protein cap GroES (Smaller heptameric subunit with 7 identiical subunits) binds.  ATP hydrolysis occur for release of GroES and the protein molecule. A  new unfolded  protein molecule can now bind to GroEL.

                                       
2.Hsp 70 (70 kDa) It is induced by stress i,e heat shock . Both eukaryotic and prokaryotic.Bind with partially folded polypeptides with exposed hydrophobic regions and prevent aggregation.

The E.coli analogs of HSP70 and Hsp40 are Dna k and Dna J respectively.

Dna J binds to unfolded protein and then to Dna k. Dna J stimulates ATP hydrolysis by Dna k. Dna-k Adp binds tightly to the unfolded proteins.
In bacteria the nucleotide exchange factor Grp E stimulates release of ADP.
ATP binds to Dna k and then the protein dissociates .


Note -Chaperones are used to prevent misfolding and aggregation .
It is necessary in crowded intracellular environment to prevent aggregation .

1.-----------------mediate protein folding .
a.Chaperones
b.Ribosomes
c.Protein itself
d.None of the above
Ans. a

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Molten globule state

Definition - It is an intermediate stage  in the protein folding pathway .(unfolded protein- molten globule -folded protein)
  
The protein folding pathaway is complicated . There are many models in support of this one of them is the hydrophobic collapsed model (molten globule) . It is called as a collapsed stage as the protein folding is initiated by a spontaneous collapse of the unfolded stage into a globular stage mediated by hydrophobic interactions.

This event is very fast (milliseceond).

Properties-

• It has most of the secondary structure in the native form.
• It is less compact.
• It lacks proper packing interactions in the interior of protein.
• The interior side remain mobile just like liquids.
• It can be measured by Circular dichroism (CD) and NMR (nuclear magnetic resonance) .
• It has a slowly fluctuating tertiary structure .
• Not specific assembly of secondary structure and the interactions.
• The initial collapsed state of a folding protein is known as a molten globule. However, a molten globule’s side chains are extensively disordered, its structure fluctuates far more than that of the native protein, and it has only marginal thermodynamic stability. 

                                         

                           Image source-https://en.wikipedia.org/wiki/Hydrophobic_collapse#/media/File:Folding_funnel_schematic.svg

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