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Measuring femto-Newton forces in enzyme turnover and its potential in highly specific pathogen detection
Daniel Mitchell1, Simona Frustaci2, Frank Vollmer2, Neil Gow3, Jennifer Littlechild1.1Henry Wellcome Building for Biocatalysis, University of Exeter, Exeter, UK. 2Life Sciences Institute, University of Exeter,
Exeter, UK.3MRC Centre for Medical Mycology, University of Exeter, UK.
Cell wall remodelling enzymes of fungal pathogens present a promising
new method of highly sensitive detection methods
Δ⍵
Δ⍵ 2
Δ⍵ 1
Δ⍵ 3
time
Enzyme conformational states
C3
C2
C1
Whispering gallery mode sensors provide single-molecule level
of detection
From this we can generate an enzymes unique activity profile, allowing us to
detect certain enzymes and substrates in a sample solution
Measuring femto-Newton forces in enzyme turnover and its potential in highly specific pathogen detection
Daniel Mitchell1, Simona Frustaci2, Frank Vollmer2, Neil Gow3, Jennifer Littlechild1.1Henry Wellcome Building for Biocatalysis, University of Exeter, Exeter, UK. 2Life Sciences Institute, University of Exeter, Exeter, UK.
3MRC Centre for Medical Mycology, University of Exeter, UK.
References:
Fungal pathogens, a world-wide health problem.Using whispering gallery mode sensors for enzyme
activity detection.
Attaching a model enzyme to the sensor. Key enzyme in pathogenic Candida albicans and
sensing
Creating a biosensor
How active is the enzyme on gold?
0 50 100 150
0.0
0.5
1.0
1.5
Concentration Serine (mM)
Acti
vit
y (
M.m
in-1
)
In Solution
On Gold
In S
olutio
n
On G
old
0.0
0.5
1.0
1.5
Vm
ax (
M.m
in-1
) In Solution
On Gold
Transaminase from
Chromobacterium violaceum
Use a transaminase enzyme from Chromobacterium
violaceum as a model test case.
R'
O
O
O
R
NH2
O
OR'
NH2
O
O
R
O
O
O
+ +
amine 1 oxo-acid 1 amine 2oxo-acid 2
• Thermophilic enzyme – stable at a
range of pH and temperatures.
• Large structural change - easier to pick
up on a sensor (solved crystal
structure).
• Relatively slow turnover – also easier to
see on sensor.
Which enzyme?
Attachment to AuNP:
AuNP Attach NTA-Ni to
AuNP Attach 6xHis Protein
We can successfully attach our enzyme to AuNPs via a 6xHis tag and it is active.
• Every year fungal infections kill approximately 1.5 million people
world-wide, yet remain understudied and underdiagnosed.1
• Multidrug resistant strains are also becoming more common.
1 G. D. Brown et. al. Science 2012; 336, pp. 647.2 https://www.gaffi.org/why/burden-of-disease-maps/cpa-prevalence.3 F. Bongomin et. al. J. Fungi, 2017; 3, pp. 57.4 M.R. Foreman et. al. Adv. Opt. Photonics, 2015; 7 pp. 168–240.
• Approximately 90% of deaths are
caused by 4 genera of fungi:
Candida, Aspergillus, Cryptococcus
and Pneumocystis.
• Detection and treatment is limited in
developing countries. Over 80 % of
deaths could be avoided with
appropriate diagnostics and
availability of antifungals.3
Estimated yearly fungal deaths in the USA2
Candida infection pathology2
Currently, the identification of fungal pathogens is largely limited to the detection of
β-1, 6-glucans on the fungal cell wall and this has serious issues with specificity
and resolution. we want to design a rapid and innovative new method for the
precise identification of fungal pathogens at low concentration.
World map showing chronic under-reporting of
fungal infections.3
Xog1 – An exoglucanase
A key enzyme of interest is the
exoglucanase, Xog1 which cleaves
the terminal glucose from glucans
within the fungi cell wall, hiding them
from our immune system.
Cleavage site
β- 1-3 glucan- Xog1 cuts the terminal glucose
Cuts any
exposed β-
1-3 glucan
Whispering galleries: optical and
acoustical
Candida albicans cell wall schematic5
The Xog1 is active and stable at a range of temperatures and attached to
gold nanoparticles making it a good choice for the biosensor!
Human antimicrobial peptide LL-37 which can bind Xog1 glucanase enzyme
1.Determine unique activity profile of Xog1 on whispering gallery sensor.
2.Human antimicrobial peptide LL-37 has been found to bind Xog16 – can use this to detect Xog1 from Candida, and bind it
to the whispering gallery sensor.
3.Use an electrochemical sensor to detect free glucose from Xog1 breakdown of β- glucans as per commonly used
diabetes sugar monitoring.7
Glucose
Glucose oxidase
FADH2
FAD
H2O2
O2
ElectrodeGluconolactone
Xog1 breaks
down β-glucans
Electrical current
5 N. A. R. Gow et. al. Nat. Rev. Microbiol. 2011; 10 pp.
112 – 122.6 P. W. Tsai et. al. PLoS One 2011; 6, e21394.7, E-H Yoo and S-Y Lee, Sensors, 2010; 10, 4558–4576.
Example detection of analyte using
antibody based whispering gallery sensor.
The femto – Newton conformational changes in enzymes during substrate
binding and turnover are impossible to measure using standard techniques.
We aim to use optoplasmonic sensors, consisting of a whispering gallery
resonator with attached plasmonic gold nanoparticle to detect the very
small changes in resonance caused by the conformational changes of an
enzyme.
From this we can build up a picture of each enzyme- substrate specific
interaction to build up a very specific single molecule detection system.
LL-37 binding site on Xog1
Δ⍵
Δ⍵ 2
Δ⍵ 1
Δ⍵ 3
time
Enzyme conformational states
C3
C2
C1
Example change in resonance frequency of the sensor when
the enzyme is in different conformations.
0 20 40 60
0.5
0.6
0.7
0.8
Concentration LL-37 (µM)
Activ
ity (µ
M.m
in-1
)
Activity of Xog1 with LL-37 Gold-labelled Xog1 on LL-37 superstructure.