To elucidate general features of structural and dynamical properties of hydration water and the influence of hydration water on the dynamical behavior of biomembranes, purple membranes from halobacteria and disk membranes from bovine retinae have been studied by neutron scattering techniques. Hydrated films of oriented multilamellar membrane stacks were used to measure lamellar diffraction patterns and quasielastic incoherent neutron scattering as a function of hydration level, of temperature, and of the protein/lipid ratio. These measurements revealed a strong interaction of a "first hydration layer" with the membrane surface and a reduced self-diffusion of aqueous solvent parallel to the membrane surface (the self-diffusion coefficient is about 5 times smaller as compared to excess water). The picosecond internal molecular motions of the protein/ lipid complex are strongly affected by the amount of solvent interacting with the lipids and the membrane proteins. In particular, the lipids and their ability to attract solvent molecules play an important role for "hydration-induced flexibility" of biomembranes. On the basis of these measurements, the impact of the hydration process on the function of biomembranes is discussed for the light-driven proton pump bacteriorhodopsin in purple membranes.

Quasielastic incoherent neutron scattering from hydrogen atoms, which are distributed nearly homogeneously in biological molecules, allows the investigation of diffusive motions occurring on the pico-to nanosecond time scale. A quasielastic incoherent neutron scattering study was performed on the integral membrane protein bacteriorhodopsin (BR), which is a light-driven proton pump in Halobacterium salinarium. BR is embedded in lipids, forming patches in the cell membrane of the organism, which are the so called purple membranes (PMs). Measurements were carried out at room temperature on oriented PM-stacks hydrated at two different levels (low hydration, h = 0.03 g of D20 per g of PM; high hydration, h = 0.28 g of D20 per g of PM) using time-of-flight spectrometers. From the measured spectra, different diffusive components were identified and analyzed with respect to the influence of hydration. This study supports the idea that a decrease in hydration results in an appreciable decrease in internal molecular flexibility of the protein structure. Because it is known from studies on the function of BR that the pump activity is reduced if the hydration level of the protein is insufficient, we conclude that the observed diffusive motions are essential for the function of this protein. A detailed analysis and classification of the different kinds of diffusive motions, predominantly occurring in PMs under physiological conditions, is presented.The main properties of a molecular machinery like an enzyme or an active transport protein are its specific architecture and internal flexibility. The study of dynamical features on a picosecond time scale reveals information on the internal flexibility, which might be correlated to the different levels of the architecture of a biological macromolecule. Fast stochastic structural fluctuations in a protein are supposed to be essential for conformational changes on a slower time scale (e.g., millisecond), which are necessary for processes like intermolecular recognition, enzymatic reactions, or other biological functions. The incoherent scattering from hydrogen atoms is dominating the total neutron scattering of biological samples (the incoherent cross section of hydrogen nuclei is '40 times larger than the cross sections of other elements) and is therefore a powerful tool for the investigation of molecular motions within a time range from 10-9 to 10-12 seconds. To a good approximation, the hydrogen atoms are distributed homogeneously in a biological macromolecule. Therefore, incoherent neutron scattering from hydrogen atoms monitors the internal motions. Early work using inelastic incoherent neutron scattering yielded dynamical information on biological systems and demonstrated the potential of this technique in the field of biophysics (1-3). Recent inelastic incoherent neutron scattering studies as well as Mossbauer spectroscopy showed that at low temperatures protein motion can largely be explained by a harmonic behavior. At temperatures between 180 and 230 K, a dynamical...

The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium. DOI: 10.1103/PhysRevLett.93.028103 PACS numbers: 87.15.He An understanding of how ligands bind to proteins is of fundamental importance in biology and medicine [1][2][3][4][5][6]. Protein:ligand association has been assumed to be dominated by factors such as the hydrophobic effect, hydrogen bonding, electrostatic, and van der Waals interactions. However, as early as 1963 it was suggested that an additional mechanism might exist, due to increased flexibility in the protein:ligand complex manifested by a change in the spectrum of vibrations due to formation in the complex of new, intermolecular interactions [7][8][9][10][11][12][13]. Theoretical normal mode analyses, used to estimate this vibrational change on insulin dimerization [12] and on water binding to bovine pancreatic trypsin inhibitor [13], have suggested that the effect is likely to be thermodynamically important. However, experimental determination of the vibrational change has been lacking.Inelastic neutron scattering, in which the dynamic structure factor Sq; ! is measured as a function of the scattering wave vector q and energy transfer h! (where ! is the angular frequency), has been used to determine the vibrational density of states (frequency distribution) g! for several proteins [14 -16]. Here we present an experimental determination of the change in g! on binding a ligand to a protein. This determination allows thermodynamic quantities associated with the vibrational change to be derived. The enzyme chosen is dihydrofolate reductase (DHFR), an important target for anticancer and antibacterial drugs [17][18][19][20][21]. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate in the presence of the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. The ligand used is methotrexate (MTX), a folate antagonist of DHFR that has been used effectively as a cytotoxic agent in the treatment of cancers [22].To minimize scattering from solvent molecules, the system was exchanged with D 2 O. To do this, lyophilized DHFR from E.coli was dissolved in D 2 O equilibrated at 4 C overnight and freeze dried. NADPH and NADPH MTX were added in equimolar ratios to the enzyme. As the dissociation constants of DHFR with NADPH and MTX are low (K For the neutron experiments, the samples were contained in an aluminum sample holder. Sample amounts were 98.1 mg (uncomplexed) and 108.6 mg (complexed). The measurements were performed on the time-of-flight spectrometer IN6 at the Institut Laue-Langevin (ILL), Grenoble, with an incident neutron beam wavelength of 5.12 Å . The scattering experiments were performed at 120 K to ensure that all dyn...

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Protein dynamics in hydrated and vacuum-dried photosystem II (PS II) membrane fragments from spinach has been investigated by quasielastic neutron scattering (QENS) in the temperature range between 5 and 300 K. Three distinct temperature ranges can be clearly distinguished by active type(s) of protein dynamics: (A) At low temperatures (T < 120 K), the protein dynamics of both dry and hydrated PS II is characterized by harmonic vibrational motions. (B) In the intermediate temperature range (120 < T < 240 K), the total mean square displacement total slightly deviates from the predicted linear behavior. The QENS data indicate that this deviation, which is virtually independent of the extent of hydration, is due to a partial onset of diffusive protein motions. (C) At temperatures above 240 K, the protein flexibility drastically changes because of the onset of diffusive (large-amplitude) protein motions. This dynamical transition is clearly hydration-dependent since it is strongly suppressed in dry PS II. The thermally activated onset of protein flexibility as monitored by QENS is found to be strictly correlated with the temperature-dependent increase of the electron transport efficiency from Q(A)(-) to QB (Garbers et al. (1998) Biochemistry 37, 11399-11404). Analogously, the freezing of protein mobility by dehydration in dry PS II appears to be responsible for the blockage of Q(A)(-) reoxidation by Q(B) at hydration values lower than 45% r.h. (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). Similar effects were observed for reactions of the water-oxidizing complex as outlined in the Discussion section.