Dynamics of proteins
Mössbauer spectroscopy
in energy and time
K. Achterhold and F.G. Parak
Physik-Department E17
Technische Universität München
James-Franck Straße, D- 85747 Garching, Germany
E-mail: Klaus.Achterhold@ph.tum.de
Fritz.Parak@ph.tum.de
http://www.physik.tu-muenchen.de/lehrstuehle/E17/
Proteins are the bricks of life. They function as enzymes, transport vehicles, storing
container and as power stations to convert light into chemical energy. These skills are
enabled by their structure and dynamics. An average structure is obtained by X-ray
crystallography or NMR spectroscopy. The Mössbauer effect allows the study of protein
dynamics. In iron containing proteins the iron is used as marker for the dynamics of the
molecule. Mössbauer spectroscopy on 57Fe has an energy resolution of 4.7 neV which
corresponds to a time sensitivity for motions faster than 140ns. Rayleigh scattering of
Mössbauer Radiation (RSMR) analysed with a Mössbauer absorber allows the
determination of an average dynamics of the whole molecule even if it contains no
Mössbauer nucleus. The density of phonons coupling to the iron can be determined by
inelastic scattering of synchrotron radiation. A 57Fe Mössbauer absorber in the scattered
beam is only in resonance if phonons deliver or absorb
the enery difference between the energy of the
synchrotron beam and the Mössbauer resonance.
An energy resolution of the synchrotron beam
of 1 meV yields a time sensitivity with a lower
limit of about 1ps.
The oxygen storing protein myoglobin serves as a
model for the investigation of the dynamics of ahelical proteins. Fig. 1 sketches the eight helices of
sperm whale metmyoglobin and the heme group with
the central iron atom.
Fig. 1
Fig. 1
Fast processes: Phonons coupled to the heme iron.
Anisotropic harmonic vibrations
a)
Phonon assisted Mössbauer effect with synchroton radiation
on a single crystal of myoglobin is used to measure the anisotropic
local vibrations of the iron. The two heme groups of the 2 molecules
within the crystallographic unit cell are shown in Fig.2a along
the c-axis and in Fig.2b along the b-axis. Blue arrows show
b)
5 orientations of the incoming beam.
Fig.3 presents some results.
Fig. 2
The left column in Fig. 3a gives the
scattering spectra for two selected orientations ( 30° and
120°). In the right column of Fig.3a the density of phonon
states is extracted from the spectra. Red and blue marks
two vibrational modes. A series of orientations can be
fitted with a cos2-law and enables to extract the direction
of vibration (Fig.3b). The vibration at 33 meV (marked in
blue) is oriented parallel to the c-axis at  =0°. The mode
at 22 meV is perpendicular to the heme.
a)
b)
Fig. 3
Achterhold, K., Keppler, C., Ostermann, A., van Bürck, U.,
Sturhahn, W., Alp, E.E. and Parak, F.G. (2002) Vibrational
dynamics of myoglobin determined by Phonon assisted
Mössbauer effect. Phys. Rev. E, 65, 051916-051911 - 051916051913.
Slow protein specific modes revealed by Mössbauer absorption spectroscopy
A Mössbauer spectrum of metmyoglobin crystals at
295K is shown in Fig.4. It can not be fitted with
Lorentzians of the typical line widts of 57Fe (dashed
line). At least one additional very broad line is
necessary.
Fig.5
<x2>[A2]
Fig.4
.08
The insets in Fig.5 demonstrate
b
.06
a
that broad lines are absent at low
temperatures.
Mean
square
.04
<x2t>
displacements, <x2>, obtained
.02
from the area of the narrow lines
<x2v> T[K]
increase linearly with temperature
0
up to 180K.
200
50
150
250
300
100
0
At higher temperatures a dramatic increase occures, and the broad lines become
measurable. We attribute this behaviour to two types of motions. The <x2v>- values are
caused by solid state like vibrations and can be calculated from the density of phonon
states or from a normal mode analysis. Above about 180K protein-specific modes start
to contribute. Due to the high energy resolution of the Mössbauer spectroscopy the
<x2t> - values stem from motions faster than 140ns (compare time resolution in Fig.4).
The broad lines indicate quasi diffusive motions. The analysis of these data together
with the results of other experiments yields the following physical picture: Protein
molecules can be in two types of states. At very low temperatures they
are frozen in a „rigid state“ (conformational substates)
where they perform harmonical motions. With increasing
Fig.6
temperature the probability increases to reach a „flexible state“
where segments of the molecules perform a Brownian
motion in limited space. It should be mentioned that only molecules in the flexible state
can perform a conformational change if e.g. the ligation changes.
Parak, F.G. (2003) Physical aspects of protein dynamics. Rep. Progr. Phys., 66, 103-129
Segmental motions determined by RSMR
collective movement:
Mössbauer
total molecule
radiation of a
a helices
57Co source is
Fig.7
Fig.8
scattered by
the sample. The scattered radiation is analysed
by a Mössbauer absorber (Fig.7). From the
inelastic scattering as function of the scattering angle one can estimate the size of the
collectively scattering segments. In myoglobin they have the size of a-helices (Fig.8).
Nienhaus, G.U., Hartmann, H., Parak, F., Heinzl, J. and Huenges, E. (1989) Angular dependent
Rayleigh scattering of Mössbauer radiation on proteins. Hyperfine Interactions, 47, 299-310