Anaerobic MicroScale Thermophoresis
Application Note NT-MO-026
Anaerobic MicroScale Thermophoresis reveals the Redox
dependency of ferredoxin in mitochondrial Fe/S biogenesis
Sven-Andreas Freibert1,2, Holger Webert1, Nina Schlinck3, Roland Lill1,2,4
1
Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Straße 6, 35032 Marburg, Germany
LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Str., 35043 Marburg, Germany
NanoTemper Technologies GmbH, Flößergasse 4, 81369 München, Germany
4
Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany
2
3
Abstract
Maturation of iron-sulphur (Fe/S) proteins
involves a complex biosynthetic machinery.
Here we investigate the interaction of one
component, Yah1, with its interaction partners
apo-Isu1 and Nfs1-Isd11. Using anaerobic
MicroScale Thermophoresis (MST) we show
the redox dependency of the interaction
between Yah1 and Isu1 as well as between
Yah1 and Nfs1-Isd11. Reduced but not
oxidized Yah1 tightly interacts with apo-Isu1
indicating a dynamic interaction between
Yah1−apo-Isu1.
Introduction
Iron-sulphur clusters (Fe/S) are one of the oldest
inorganic prosthetic groups on earth. They play
important roles in fundamental cellular processes
such as respiration, amino acid and cofactor
metabolism, DNA metabolism and repair, and the
regulation of environmental responses 2-4. The
mitochondrial ISC (iron-sulphur cluster assembly)
machinery is crucial for the synthesis of all Fe/S
clusters (mitochondrial, cytosolic and nuclear) as
well as for cellular iron regulation. Mutations in
many
mitochondrial
ISC
genes
cause
neurodegenerative, haematological and metabolic
diseases with complex clinical and biochemical
phenotypes 5,6-8.
Figure 1: NMR solution structure of the yeast ferredoxin
Yah1 (PDB: 2MJE), in its reduced state and with its flexible Cterminus. The [2Fe-2S] cluster is represented as stick model.
Isu1-interacting residues are in sphere-representation.
The first step of Fe/S protein maturation in yeast
crucially requires the scaffold protein Isu1, the
cysteine desulfurase complex Nfs1-Isd11 9 and
the [2Fe-2S] ferredoxin Yah1 (Fig. 1) 10-14. So far,
the exact nature of the interactions within the
machinery has not been resolved in detail.
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Results
Reduced mitochondrial ferredoxin Yah1
interacts with Isu1
The physiological interaction partner of Yah1
within the mitochondrial ISC assembly machinery
has remained elusive. Yah1 is predicted to interact
in its reduced state with Isu1 to productively
deliver electrons for Fe/S cluster synthesis.
Because determining a Kd anaerobically via
equilibrium titration has only been possible since
MicroScale Thermophoresis was launched, the
influence of the Yah1 redox state for Isu1
interaction was addressed by this novel technique
the first time 15.
a
Increasing amounts of reduced or oxidized Yah1
were mixed with fluorescently labelled Isu1 under
anaerobic conditions and the thermophoretic
behaviour was followed using wax-sealed
capillaries on a Monolith NT.115. Isu1 strongly
interacted with reduced Yah1 (Kd = 25 nM), yet
100-fold weaker with oxidized Yah1 (Fig. 2a). This
result made it possible to determine the interaction
interface on Yah1 via NMR (Fig. 1).
b
Isu1+Yah1-Ox
Isu1+Yah1-Red
3000
Kd [nM]
2500
2000
Kd = 25.5 nM
1500
Kd = 2.4 µM
1000
500
0
c
Nfs1-Isd11+Yah1-Red
Kd = 2.5 µM
Nfs1-Isd11+Yah1-Ox
Kd = 240 nM
Figure 2: Quantitative determination of the interaction between the scaffold protein Isu1 or the cysteine desulfurase complex
Nfs1-Isd11 with Yah1. (a) Bar chart showing Kd. Isu1 preferentially interacts with reduced Yah1 while Nfs1-Isd11 complex better
interacts with oxidized Yah1. Yah1 was used as isolated (Yah1(ox)) or reduced with dithionite (Yah1(red)), and mixed at increasing
concentrations with 200 nM fluorescently labeled Isu1 or Nfs1-Isd11. MicroScale Thermophoresis experiments were performed, and
dissociation constants (Kd) were determined (Error bars indicate the SD (n=6). (b) Isu1 or Nfs-Isd11 was labelled with the fluorescent
dye NT 647 (NanoTemper Technologies, Munich Germany). All steps were performed under anaerobic conditions. Yah1 was reduced
with sodium dithionite at a 5-fold molar excess. After incubation for 25 min dithionite was removed using Zeba Spin desalting columns
(Thermo Scientific). Fluorescently labelled Isu1 or Nfs1/Isd11 (final concentration of 200 nM) was added to the indicated
concentrations of Yah1. The MST binding reactions were measured using Monolith NT.115 at 32 °C or 28 °C for Isu1-Yah1 and Nfs1Isd11-Yah1, respectively. (LED power: 100 %, MST power: 80 %). The Kd values were estimated from at least six independent
experiments using NanoTemper Analysis 1.5.37 and Origin 8G software.
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All titrations reached saturation and sigmoidal
curves could be fitted (Fig. 2b). The results
demonstrate a preferential interaction of Isu1 with
reduced Yah1, thus facilitating electron transfer
during de novo Fe/S cluster synthesis. We further
used anaerobic MicroScale Thermophoresis to
investigate the potential interaction between Yah1
and Nfs1, since the bacterial counterparts were
reported to form a complex 16. Reduced Yah1
showed only a weak interaction with Nfs1-Isd11, in
the Kd range of the complex of oxidized Yah1 and
Isu1 (Fig. 2a, c). Surprisingly, a tenfold higher
affinity was observed for oxidized Yah1 and Nfs1Isd11 (Fig. 2a, c), suggesting a dynamic
rearrangement of Yah1 within the ISC biosynthetic
complex after electron transfer. Notably, the
binding between reduced Yah1 and apo-Isu1 was
still 10-fold stronger than that between oxidized
Yah1
and
Nfs1-Isd11,
emphasizing
the
physiological relevance of reduced Yah1−apoIsu1 interaction for initiating Fe/S cluster
synthesis.
Discussion
MicroScale Thermophoresis allowed anaerobic
equilibrium titrations, which has never been
possible before. This method identified a strong
interaction (Kd = 25 nM) between reduced Yah1
and apo-Isu1. A 100-fold weaker affinity was
observed for oxidized Yah1 and apo-Isu1
(Δ(ΔG)Ox/Red = 2.9 kcal/mol). The high preference
of apo-Isu1 for the reduced form of Yah1 provides
strong evidence for the physiologically relevant
encounter of the two proteins. Additionally,
anaerobic thermophoresis revealed a higher
affinity of oxidized Yah1 to Nfs1-Isd11 than to
Isu1, suggesting that upon oxidation of Yah1 it
loosens the interaction with Isu1 and undergoes
tighter binding with Nfs1-Isd11. These results
suggest a so far undescribed dynamic
rearrangement of the five-membered ISC complex
during Fe/S cluster formation.
Methods
Affinity
measurements
using
anaerobic
MicroScale Thermophoresis (MST). MST was
performed on a Monolith NT.115 (NanoTemper
Technologies GmbH, Munich, Germany) at 32 °C
or 28 °C (LED power (red channel) was set
between 60 % and 100 % and MST power to
80 %) 33. All samples and dilution series were
prepared anaerobically in a COY anaerobic
chamber (95 % N2; 5 % H2). Samples were filled in
the capillaries and wax-sealed prior to
measurement. Isu1 or Nfs1-Isd11 (20 µM each)
were labeled with the dye NT647 according to the
supplier (NanoTemper Technologies). 200 nM
Isu1 or Nfs1-Isd11 were titrated with unlabeled
Yah1, FDX2, FDX1 or Yfh1 in 35 mM KPi pH 7.4,
150 mM NaCl. At least six independent MST
experiments were recorded at 680 nm and
processed by NanoTemper Analysis 1.2.009 and
Origin8.
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