“Gütlich, Bill, Trautwein: Mössbauer Spectroscopy and Transition Metal Chemistry@Springer-Verlag 2009”

Dynamics of proteins Mössbauer spectroscopy

in energy and time

K. Achterhold and F.G. Parak

Physik-Department E17Technische Universität München

James-Franck Straße, D- 85747 Garching, GermanyE-mail: Klaus.Achterhold@ph.tum.de

Fritz.Parak@ph.tum.dehttp://www.physik.tu-muenchen.de/lehrstuehle/E17/

“Gütlich, Bill, Trautwein: Mössbauer Spectroscopy and Transition Metal Chemistry@Springer-Verlag 2009”

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 absorbthe 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 -helical proteins. Fig. 1 sketches the eight helices of sperm whale metmyoglobin and the heme group with the central iron atom.

Fig. 1Fig. 1

“Gütlich, Bill, Trautwein: Mössbauer Spectroscopy and Transition Metal Chemistry@Springer-Verlag 2009”

Fig. 2

a)

b)

Fast processes: Phonons coupled to the heme iron.Anisotropic harmonic vibrations

Phonon assisted Mössbauer effect with synchroton radiation on a single crystal of myoglobin is used to measure the anisotropiclocal vibrations of the iron. The two heme groups of the 2 moleculeswithin the crystallographic unit cell are shown in Fig.2a along the c-axis and in Fig.2b along the b-axis. Blue arrows show5 orientations of the incoming beam.

Fig. 3

a)

b)

Fig.3 presents some results.The left column in Fig. 3a gives thescattering 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.

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 - 051916-051913.

“Gütlich, Bill, Trautwein: Mössbauer Spectroscopy and Transition Metal Chemistry@Springer-Verlag 2009”

Slow protein specific modes revealed by Mössbauer absorption spectroscopy

Fig.4 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.

0 50 100 150 200 250 3000

.02

.04

.06

.08

<x2 >[A

2 ]T[K]

<x2t>

<x2v>

abThe insets in Fig.5 demonstrate

that broad lines are absent at low temperatures. Mean square displacements, <x2>, obtained from the area of the narrow lines increase linearly with temperature up to 180K. At higher temperatures a dramatic increase occures, and the broad lines become measurable. We attribute this behaviour to two types of motions. The <x2

v>- 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 <x2

t> - values stem from motions faster than 140ns (compare time resolution in Fig.4).

Fig.5

“Gütlich, Bill, Trautwein: Mössbauer Spectroscopy and Transition Metal Chemistry@Springer-Verlag 2009”

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

Fig.6

are frozen in a „rigid state“ (conformational substates)where they perform harmonical motions. With increasing temperature the probability increases to reach a „flexible state“ where segments of the molecules perform a Brownianmotion 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

Fig.7

Mössbauer radiation of a 57Co source is 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 -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

collective movement:

total molecule

helices

Fig.8