Analysis of Peptidoglycan Flashcards by Daniel Goff

Difference between cell envelope and cell wall?

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

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

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

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

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Structure of Peptidoglycan building block (draw/explain)

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

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

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

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

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

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

Highly conserved

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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?

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

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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?

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

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Do organisms only contain 1 type of cross-link?

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

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

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

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Old school representation of peptidoglycan layer
More accurate, newer representation

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

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

Cleaved to tetrapeptide

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Draw 4-3 crosslink

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What amino acid is Q and what is E?

Q - Glutamine
E - Glutamic acid

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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?

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

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

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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?

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

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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?

(see word document for structure)

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

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

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

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

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

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

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?

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

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

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)

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

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)

What does retention time of PG fragments reflect?

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

What does composition of multimers reflect?

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

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

Used as an acceptor for transpeptidation

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

Mass spectrometry provides information related to mass of sample Mass spectrometry doesn’t directly measure mass

Explain MALDI method of mass spec.

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

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

Spray of ions through detector Acidic conditions forms positives ions - Binds e.g. amines Basic conditions forms negative ions - Binds e.g. carboxylic acids

Mass spectrometry definition

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

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

Monoisotopic mass vs average mass Ion charge

Monoisotopic mass vs average mass

Monoisotopic mass – Mass of a molecule exclusively made of most abundant isotopes Average mass – Mass of a molecule containing a mixture of all isotopes

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

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

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)

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

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

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

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

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

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

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

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

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?

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

Go through case study exercises of (2)

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

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+

Is E. coli Gram +ve or -ve

Gram -ve

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

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