Introduction, Motivation

Domain movements, conformational changes and protein dynamics play a crucial role in the catalytic
reaction of enzymes. Highly diverse structural changes have been described, and in order to obtain a
comprehensive understanding of the functional role of these structural motifs and rearrangements
the movements need to be visualized. Here we suggest setting up for the first time small infrared
probes in membrane proteins from the respiratory chain.

Domain movements, conformational changes and protein dynamics play a crucial role in the catalytic
reaction of enzymes. Highly diverse structural changes have been described, including include rotor
like movements (Fo-F1-ATPase), drilling of helices (TIM/TOM), the switching of subunits between two
sites (respiratory complex III or K+-ATPase) or wavelike motions in transporters.

In order to obtain a comprehensive understanding of the functional role of these structural motifs
and rearrangements on molecular level and in order to determine the specific structural
environment required the movements needs to be visualized. Several of these conformational
movements (for example in ATPase) have been previously identified by large probes typically used
for fluorescence or EPR spectroscopies. Here we suggest setting up infrared probes that are
significantly smaller and thus less perturbative for the structures and the catalytic activity.

IR probes such as nitrile- and thiocyanate-derivatized amino acids have been first described for
peptides and soluble proteins (Farfarman et al. 2006, JACS). They have been found to give specific
information on the local environment of the probe, because their IR absorption frequencies strongly
depend on the hydrogen bonding with the surrounding protic solvent molecules, backbone, or amino
acids.

Figure 1: Scheme of a –CN IR probe introduced at three different positions of a helix, with distinct
structural and electro-static
environments (A) and position
of the CN vibration in different
water/THF mixtures (B)
(spectra adapted from Bishak
et al. (2010) Biophysical
Journal).

 In this project we suggest introducing these probes in large membrane proteins from the
respiratory chain and to target large domain movements that rule the catalytic mechanisms.

 The protocol established will then be adapted to further proteins, as domain movements are
also crucial in other processes like for example signaling, allosteric changes, polymerization
reactions, electron proton or ion transfer.

 The changes in the spectral signature induced by electron transfer or ligand binding will then
be monitored by reaction induced vibrational spectroscopies. It is noted that reaction
 induced difference spectroscopy enables the study of the reaction of the protein without
contributions of solvent. Information on the reorganization of the backbone, cofactors and of
individual amino acids can be depicted in this type of data and correspondingly the
reorganization of the introduced IR label upon the induced reaction.

2. Labelling and sample preparation

This interdisciplinary project includes the preparation of the labeled samples, a biochemical analysis
to exempt an eventual perturbation of the catalytic activity and then the analysis by infrared and
Raman spectroscopies. For the preparation of the labeled sample the key step is the native chemical
ligation that involves an initial transthioesterification reaction followed by intramolecular S->N acyl
shift to generate an amide bond. Cysteine free proteins have been produced and cysteine groups at
selected positions have already been introduced at critical positions at the protein in the partner labs
in Germany (Prof. Thorsten Friedrich, University of Freiburg and Prof. Michael Boersch, University
clinics Jena). First labeling attempts on cysteine mutants have been successfully made by a master
student in the lab in Strasbourg. Protein purification and biochemical analysis will be made in the
experienced biochemistry laboratories in Freiburg and Jena; the student will visit the labs in order to
learn these procedures. As the group in Strasbourg has a well-established expertise in reaction
induced FTIR and Raman spectroscopies, this part of the project will be realized in the group in
Strasbourg.

3. Proteins studied

3.1. The piston like movement of respiratory complex I. The first part of the study concerns the
reorganization of the amphipathic helix in complex I The respiratory complex I, is the entry point for
electrons into most respiratory chains generating the proton motive force required for cellular
energy consuming processes. Sequence analysis revealed that complex I evolved from pre-existing
modules for electron transfer and proton-translocation (Friedrich and Scheide, 2000, Biochemistry).
The three largest subunits in the membrane arm, NuoL, NuoM and NuoN, show primary sequence
similarity to one particular class of antiporters, and are thus predicted to play a role in the proton
translocation machinery. Recent crystallographic data revealed the presence of a unique helix of
140 Å length within the membrane arm of the complex (Baradaran et al. 2013, Nature). The
working hypothesis is that complex I couples electron transfer with proton translocation by
controlling the conformational state of the helix.

 We aim introducing -CN labels on critical positions of this helix and visualize the movement
during upon electron transfer and also in the presence of the natural substrates (NADH,
ubiquinone) and inhibitors to the proton path (Zn2+). The studies on complex I will be
performed in collaboration with the laboratory in Freiburg. So far, the following variants,
located at important positions in the membrane were created for spin labeling using the
established expression and purification system: R529C, K551C, P552C, F553C, L554C, L560C,
K561C, R562C, N566C, I571C, P572C, A573C, V574C, Y590C The same mutants will now be
coupled to the IR probe.
Figure 2 shows the amphipatic helix (magenta) that links the antiporter units (adapted from Efremov
and Sazanov, (2011), Nature).

It seems self-evident that a possible movement of the helix would transmit the energy released by
the electron transfer reaction with proton translocation, but there is no experimental evidence.

3.2. Spotlighting motors and controls of single FoF1-ATP synthase. The second part of the project
concerns the ATP synthase, a well know rotary engine that produces most of the adenosine-5′-
triphosphate (ATP) in living cells. The ubiquitous multi-subunit enzyme is located in the plasma
membrane of bacteria, the thylakoid membrane in photosynthetic cells and in the inner
mitochondrial membrane of eukaryotes. The enzyme operates as a mechanochemical energy
transducer comprising two motors with different step sizes.

Here we suggest giving evidence for the participation of the -subunit to the ATP production (shown
in pink in Figure 3). This subunit was found in two different conformations depending on
experimental conditions; however, no direct evidence exists for this. The small -CN label described
above is able to probe this movement without perturbing the kinetics and the structural
properties. The studies will be performed in collaboration with the group of Prof. M. Börsch in Jena.
WT and cysteine mutants at the C-terminus are already available in this group.

Figure 3: rotary subunit movements of FoF1-ATP synthase and ε conformational changes (Börsch and
Duncan, 2013, Biochem. Soc. Trans ).