F. Parak

Hydrogen and deuterium in myoglobin as seen by a neutron structure determination at 1.5 Å resolution

From the first days of protein neutron structure determination sperm whale myoglobin was an object under investigation [Nature 224 (1969) 143, J. Mol. Biol. 220 (1991) 381]. Nevertheless myoglobin is still of interest [Proc. Natl. Acad. Sci. USA 97 (2000) 3872]. The feasibility of the monochromatic neutron diffractometer BIX-3 at the JRR-3M reactor at the JAERI [J. Phys. Chem. Solids 60 (1999) 1623], to collect high-resolution diffraction data in a relatively short time stimulated us to repeat the structural determination of myoglobin. The structure of metmyoglobin has been determined up to a resolution of 1.5 A. The hydrogen atoms were replaced in part, by deuterium soaking the crystals for more than 10 years in D(2)O. A refinement of all atoms has been performed including the refinement of individual mean square displacements and occupancies of the exchangeable protons in backbone hydrogen bonds. A method is described to show clear negative scattering densities of the H atoms. Water molecules within the protein and on the molecule surface are shown. The exchangeability of H atoms is correlated with structural distribution and flexibility.

Correlation between protein flexibility and electron transfer from QA-* to QB in PSII membrane fragments from spinach.

To analyze a possible correlation between the extent of QA-* reoxidation and protein dynamics, fluorometric and Mössbauer spectroscopic measurements were performed in photosystem II membrane fragments from spinach. Numerical evaluation of the flash-induced change of the normalized fluorescence quantum yield revealed that the extent of reoxidation starts to decrease below 275 K and is almost completely suppressed at 230 K. Detailed analyses of Mössbauer spectra measured at different temperatures in 57Fe-enriched material indicate that the onset of fluctuations between conformational substates of the protein matrix occurs also at around 230 K. Based on this correspondence, protein flexibility is inferred to play a key role for QA-* reoxidation in photosystem II. Taking into account the striking similarities with purple bacteria and the latest structural information on these reaction centers [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816], it appears most plausible that also the headgroup of plastoquinone-9 bound to the QB-site in PSII requires a structural reorientation for its reduction to the semiquinone.

Hydrogen atoms in proteins: Positions and dynamics

Hydrogen atoms constitute about half of the atoms in proteins. Thus they contribute to the complex energy landscape of proteins [Frauenfelder, H., Sligar, S. G. & Wolynes, P. G. (1991) Science 254, 1598-1603]. Neutron crystal structure analysis was used to study the positions and mean-square displacements of hydrogen in myoglobin. A test of the reliability of calculated hydrogen atom coordinates by a comparison with our experimental results has been carried out. The result shows that >70% of the coordinates for hydrogen atoms that have a degree of freedom is predicted worse than 0.2 Å. It is shown that the mean-square displacements of the hydrogen atoms obtained from the Debye-Waller factor can be divided into three classes. A comparison with the dynamic mean-square displacements calculated from the elastic intensities obtained from incoherent neutron scattering [Doster, W., Cusack, S. & Petry, W. (1989) Nature 337, 754-756] shows that mainly the side-chain hydrogen atoms contribute to dynamic displacements on a time scale faster than 100 ps.

Protein structural dynamics as determined by Mössbauer spectroscopy

Mössbauer spectroscopy on57Fe allows the study of dynamics with a characteristic time faster 100 ns. For myoglobin a detailed physical picture of protein dynamics has been obtained. A myoglobin molecule has no well defined energy minimum. X-ray structure analysis yields only an average conformation. At low temperatures the molecules are trapped in slightly different structures called conformational substates. At higher temperatures a Brownian type of oscillation of molecular segments in restricted space occurs. RSMR technique allows an estimation of the characteristic size of these segments which are in myoglobin well below 30 A and larger than 6 A. A determination of the quasielastic absorption with high accuracy yields the energy distribution of the conformational substates. As further examples bacteriorhodopsin and a model compound for membranes are discussed.

The hydration shell of myoglobin

The space in the unit cell of a metmyoglobin crystal not occupied by myoglobin atoms was filled with water using Monte Carlo calculations. Independent calculations with different amounts of water have been performed. Structure factors were calculated using the water coordinates thus obtained and the known coordinates of the myoglobin atoms. A comparison with experimental structure factors showed that both the low and the high resolution regime could be well reproduced with 814 Monte Carlo water molecules per unit cell with a B-value of 50 Å2. The Monte Carlo water molecules yield a smaller standard R-value (0.166) than using a homogeneous electron density for the simulation of the crystal water (R = 0.212). A reciprocal space refinement of the water and the protein coordinates has been performed. Monte Carlo calculations can be used to obtain information for crystallographically invisible parts of the unit cell and yield better coordinates for the visible part in the refinement.

Protein dynamics: determination of anisotropic vibrations at the haem iron of myoglobin

The phonon assisted Mossbauer effect is used to determine the anisotropic harmonic vibrations labelled by the iron in the active centre of myoglobin at room temperature. A single crystal of metmyoglobin is investigated in five different orientations. Several modes are assigned by the projection of the vibrational amplitude onto the beam directions. The density of phonons below 1 meV shows a quadratic increase with the energy like that in a Debye solid. An anisotropic velocity of sound in the protein crystal is extracted with a mean sound velocity of 1657 m s−1. Modes between 4 and 5 meV are identified as haem sliding motions. Vibrations between 30.2 and 36.5 meV are mainly within the haem plane; those between 19 and 25.6 meV are perpendicular to the plane. These results together with phonon assisted Mossbauer effect measurements on hydrated myoglobin powder and polycrystal samples in the temperature range between 50 K and room temperature are compared with the results of Mossbauer absorption experiments. While the mean square displacements at the iron obtained by the phonon assisted Mossbauer effect increase linearly with temperature up to room temperature, Mossbauer absorption reveals a dynamical transition temperature. Above this temperature a much stronger increase of the mean square displacements occurs, indicating protein specific and functionally important dynamics. Since these displacements are not registered by the phonon assisted Mossbauer effect the involved energy transfer is beyond the energy resolution of the method i.e. smaller than 1 meV. Actually the energy transfer is in the nanoelectronvolt energy regime as seen from the energy profile of the Mossbauer absorption spectrum. Protein specific dynamics can be explained as diffusive motion of molecular segments in limited space.

Dynamics of protein-water systems revealed by Rayleigh scattering of Mössbauer radiation (RSMR)

A critical review of recent studies of protein dynamics by the RSMR technique is given. The main approximations in quantitative analyses of RSMR data are discussed and conclusions about dynamical properties of protein and interprotein water, deduced from experiments, are described.

Glass-like behaviour of proteins as seen by Mössbauer spectroscopy

Abstract Mossbauer spectroscopy reveals a strong analogy in the dynamic properties of 57 Fe in glycerol and in myoglobin crystals. Above about 200 K, the shape and the area of the spectra can be explained by a jump diffusion model with a jump time distribution according to Cole-Davidson. In the high-viscosity region well below 200 K, the myoglobin molecules have a similar but not an identical structure indicating a highly degenerated ground state energy of the molecules.

The mobility of chromatophore membranes from Ectothiorhodospira Shaposhnikovii revealed by Rayleigh scattering of Mössbauer radiation (RSMR) experiments

AbstractChromatophores from Ectothiorhodospira Shaposhnikovii in solvents of different viscosity were investigated by RSMR experiments in the temperature range between 112 K and room temperature. Additional RSMR-experiments were done on solvents only. The mobility of the molecules and within the molecules is the given by the Debye-Waller factor which yields the meansquare displacement,