Analysis of Peptidoglycan

Question 1

Difference between cell envelope and cell wall?

Answer 1

Cell wall includes peptidoglycan and molecules associated with it (e.g. proteins, (lipo)teichoic acids, sugars)
Cell envelope is that + cytoplasmic membrane

Question 2

Gram +ve vs Gram -ve cell envelope arrangement
Another (more reliable) classification name for both?

Answer 2

Gram +ve has large peptidoglycan layer
Gram -ve has outer and inner membrane with peptidoglycan in between

Monoderm (gram +ve) and Diderm (gram -ve)

Question 3

Structure of Peptidoglycan building block (draw/explain)

Answer 3

Alternating GlcNAc and MurNAc bound by 1,4 glycosidic bond
MurNAc has L- and D- (penta)peptide chain

Question 4

Structure of pentapeptide stem in gram +ve (draw/explain)
What extra features can be present and what enables this?

Answer 4

L-Ala - Glutamine (Q) - L-Lys - D-Ala - D-Ala

Di-AA (amino acid) allows for lateral chain or polymerisation
- One amino group to form peptide bond; Another to cross-link and allow polymerisation

Question 5

Structure of pentapeptide stem in gram -ve (draw/explain)

Answer 5

L-Ala - Glutamic acid (E) - mDap - D-Ala - D-Ala

Question 6

Within a species of bacteria, how can the pentapeptide stem be described?

Answer 6

Highly conserved

Question 7

What does D,D transpeptidation recognise and what happens?
What is it mediated by?
What bonds does it form?
What happens to donor stem during cross-linking?

Answer 7

D,D transpeptidases recognise D-Ala - D-Ala extremity of donor and from a cross-link between 4th position D-Ala with L-Lys of acceptor

Mediated by Penicillin Binding Proteins (PBPs)

Forms 4-3 bonds

Terminal D-Ala is cleaved from donor to make it a tetrapeptide

Question 8

What does L,D transpeptidation recognise and what happens?
What do the enzymes that it works with do? (hint - trimming and substrate)
What bonds does it form?

Answer 8

Recognises L-Lys - D-Ala of donor and forms a cross-link between L-Lys of donor and L-Lys of acceptor

Enzymes trim terminal D-Ala to give tetrapeptide so L-Lys - D-Ala is exposed
Enzymes then use position 4 D-Ala as substrate and then cleave it

Forms 3-3 bonds

Question 9

Do organisms only contain 1 type of cross-link?

Answer 9

No they can have 4-3 and 3-3 bonds

Question 10

How does β-lactam (penicillin) target D,D transpeptidases?
How is resistance to this developing in bacteria?

Answer 10

Mimics D-Ala – D-Ala extremity
β-lactam ineffective towards L,D transpeptidases as it recognises different part of stem; More bacteria using this type of cross-link

Question 11

Old school representation of peptidoglycan layer
More accurate, newer representation

Answer 11

Rigid exoskeleton (protective role)
Acts as a sieve to regulate exchanges with surrounding environment

Dynamic 3D network with elasticity and plasticity; Not rigid or crystalline
Subcellular compartment
Dynamic regulation of exchanges with the environment

Question 12

What has happened to pentapeptide donor stem after cross-linking?

Answer 12

Cleaved to tetrapeptide

Question 13

Draw 4-3 crosslink

Question 14

What amino acid is Q and what is E?

Answer 14

Q - Glutamine
E - Glutamic acid

Question 15

Acid hydrolysis of a PG sample extracted from an unknown bacterium revealed the presence of GlcNAc, MurNAC, Alanine, Glutamine and Lysine in a molar ratio of 1:1:3:1:1
- Can you deduce the composition of the PG building block of this bacterium?
- Do you think it is likely to be a Gram-positive or a Gram-negative organism? Why?

Answer 15

GlcNAc-MurNAc-Alanine-Glutamine (Q)-Lysine-DiAlanine

Gram positive as it contains glutamine (pos.2), lysine (pos.3)

Question 16

Acid hydrolysis of a purified PG fragment resulting from enzymatic digestion revealed the presence of GlcNAc, MurNAc, Alanine, Glutamine and Lysine in a molar ratio of 2:2:3:2:2
- What could be the structure(s) of this PG fragment? Is one structure more likely? Why?
- What are the enzymatic activities that are required to generate this muropeptide?

Answer 16

Could have Alanine as a lateral chain, however with this ratio only one could have the lateral chain; Unlikely as the chain is highly conserved, so if one has it then all must have it
Certain structures are less likely as they would only result form partial enzymatic digestion
(see word document for structure)

The enzymatic activities required to generate this muropeptide must cleave the DiAlanine from the acceptor molecule

Question 17

Acid hydrolysis of a purified PG fragment revealed the presence of GlcNAc, MurNAc, Alanine, Glutamine and Lysine in a molar ratio of 1:1:4:1:1
- Can you propose a structure for this PG fragment?

Answer 17

(see word document for structure)

Question 18

What are the 3 steps required for preparation of peptidoglycan fragments for structural analysis

Answer 18

PG purification
Digestion with hydrolytic enzymes as molecule is too big and insoluble
PG reduction (sodium borohydride)

Question 19

Key steps involved in peptidoglycan purification (5 key steps; Not including wash steps)

Answer 19

Grow cells till exponential phase
Break them up
Boil for 30 mins
Wash in distilled water
Treat with proteases
Wash
Treat with HCl to remove molecules associated with peptidoglycan through phosphodiester bonds (e.g. TA, capsules)
Wash

Question 20

Why are lysozymes used for PG digestion and what does digestion allow?
What is used on stronger bacteria resistant to lysozyme?

Answer 20

They are muramidases; Cleave after MurNAc to leave just GlcNAc-MurNAc
This allows for solubilisation of PG fragment

Mutanolysin is used as its a stronger lysozyme

Question 21

What do PG monomers in solution exist as?
How does this occur?
Why is this a problem for the chromatogram from rp-HPLC?
What molecule is used for reduction and how does this help?

Answer 21

Equilibrium mixtures of the straight and cyclic forms

Formation of a pyranose ring (C6) results from intramolecular nucleophilic attack of one of the OH’s on the carbonyl C of the aldehyde
On nucleophilic attack to form the ring, the carbonyl O becomes an OH which points either below the ring (α anomer) or above the ring (β anomer); On C1 of top right ring

During rp-HPLC separation, the α and β anomers (stereoisomers) appear as 2 peaks

Reduction with NaBH4 simplifies the chromatogram by keeping it in straight form

Question 22

What step does rp-HPLC follow?
What does rp-HPLC stand for?
How is it different to normal phase-HPLC?

Answer 22

Follows PG digestion into Disaccharide-peptides

Reverse Phase-High Pressure Liquid Chromatography

rp-HPLC stationary phase is hydrophobic; Makes more sense for PG fragment
Elution of hydrophobic molecules bond by decreasing the polarity of the mobile phase (use organic solvents)

Question 23

Explain rp-HPLC chromatogram (hint - absorbance and retention times)

Answer 23

The greater the absorbance, the greater the abundance
As size increases, you lose sensitivity

For retention/elution time, the more complex a molecule is, the more it will interact with the resin (greater retention time)

Question 24

What does retention time of PG fragments reflect?

Answer 24

Increasing complexity = Higher retention times

e.g. Within monomeric structures, ones which have longer lateral chain have longer retention time
e.g. Between monomeric and dimeric, there is a large difference in retention time

Difference in retention time means it will form different peaks

Question 25

What does composition of multimers reflect?

Answer 25

Composition of monomers
Get symmetrical repetition of the basic monomer
To get from dimer to trimer, you can only build upon this using what is available i.e. monomer

Question 26

The most abundant monomeric structure is the most likely to be what?
Go through exercises 1-2 in rp-HPLC section of (1)

Answer 26

Used as an acceptor for transpeptidation

Question 27

What information does mass spectrometry provide?
What does it not give?

Answer 27

Mass spectrometry provides information related to mass of sample

Mass spectrometry doesn’t directly measure mass

Question 28

Explain MALDI method of mass spec.

Answer 28

The sample is mixed with a matrix and spotted on a metal plate

Once dried, desorption is triggered by a UV laser beam

Ionisation occurs at this stage, and you measure time taken for ion to fly through system before it hits detector

Question 29

Explain Electro spray ionisation (ESI)
Why is HPLC of sample carried out in either acidic or basic conditions?
What may these bind?

Answer 29

Spray of ions through detector

Acidic conditions forms positives ions - Binds e.g. amines
Basic conditions forms negative ions - Binds e.g. carboxylic acids

Question 30

Mass spectrometry definition

Answer 30

Mass spectrometry is an indirect analytical method used to determine the mass and composition of molecules by measuring their mass-to-charge ratio (m/z) after ionisation

Question 31

What 2 things must be considered when analysing mass spec data?

Answer 31

Monoisotopic mass vs average mass
Ion charge

Question 32

Monoisotopic mass vs average mass

Answer 32

Monoisotopic mass – Mass of a molecule exclusively made of most abundant isotopes

Average mass – Mass of a molecule containing a mixture of all isotopes

Question 33

What are the 2 ways the same molecule can generate several ions of different isotopic variants?

Answer 33

Ionisation state
Type of adduct ion (H+, Na+ etc.)

Question 34

MS analysis of a PURE peptide reveals the presence of a singly charged ion with an m/z value of 1099.469
What is the monoisotopic mass of the corresponding molecule?
(proton mass = 1.0078)

Answer 34

1099.469 x 1 = 1099.469; 1099.469 – 1.0078 = 1098.461 Da

Question 35

Calculate m/z of a both singly and doubly charged ions of a molecule with monoisotopic mass of 1223.723 Da

Answer 35

Singly charged ion:
(noted [M+H]1+) has a m/z = (1223.713 +1.0078)/1 = 1224.7208

Doubly charged ion:
(noted [M+2H]2+) has a m/z = (1223.713 +(2x1.0078)/2 = 612.8693

Question 36

What is an adduct?
Can you get adducts with combinations of different ions? Give examples

Answer 36

Product that forms when 2 or more distinct molecules bond together

Yes; e.g. [M+Na+H]2+, [M+Na+K]2+, [M+2H+Na]3+
As number of charges increase, number of combinations also increases

Question 37

Go through exercises for calculating m/z of different adducts in (2)

Question 38

What does spacing between ion peaks tell us about charge state?
Equation for ∆m
(look at image on (2) if needed

Answer 38

The smaller the spacing, the higher the charge
∆m = 1 in singly charged
∆m = 0.5 in doubly charged

∆m = 1/z(charge)

Question 39

What is Tandem mass spec (MS/MS)?
Where is the most common cleavage site and what are the most common ‘series’ formed?
What is the mass difference between 2 adjacent b or y ions indicative of?
How do you reconstitute overall structure?

Answer 39

When a molecule is fragmented to form several fragment ions

Commonly cleaved at CO-NH bond and forms b or y series

Mass difference indicative of a particular amino acid

Identify m/z values of the different parts and you can reconstitute overall structure

Question 40

Go through case study exercises of (2)

Question 41

How can predicted PG structure and mass spec data be combined?

Answer 41

Calculate mass of predicted structure of see if it aligns with monoisotopic mass we calculated from mass spec m/z
- Consider if its reduced as this adds an extra 2H+

Question 42

Is E. coli Gram +ve or -ve

Answer 42

Gram -ve

Question 43

What do you need to consider when calculating mass of predicted PG structure?

Answer 43

Mass of water being lost as peptides and sugars added in condensation reactions