Direct measurement of hydration-related dynamic changes in lysozyme using inelastic neutron scattering spectroscopy

Inelastic neutron scattering spectroscopy is used to investigate dynamic changes in lysozyme powder at two different low D2O hydrations (0.07g D2O/g protein and 0.20 g D2O/g protein). In the higher hydration sample, the inelastic scattering between 0.8 and 4.0 cm-1 energy transfer is increased and the elastic scattering is decreased. The decreased elastic scattering suggests increased atomic amplitudes of motion and the increased 0.8 to 4.0 cm-1 scattering suggests increased motions in this frequency range. Comparison with normal mode models of lysozyme dynamics shows that the inelastic difference occurs in the frequency region predicted for the lowest frequency, largest amplitude, global modes of the molecular [M. Levitt, C. Sander and P.S. Stern, J. Mol. Biol. 181, 423 (1985). B. Brooks and M. Karplus, Proc. Natl. Acad. Sci (U.S.A) 82, 4995 (1985), R.E. Bruccoleri, M. Karplus and J.A. McCammon, Biopolymers 25 1767 (1986)]. Our results are consistent with a model in which an increased number of low frequency global modes are present in the higher hydrated sample.     

Hydration effect on low-frequency protein dynamics observed in simulated neutron scattering spectra

Hydration effects on protein dynamics were investigated by comparing the frequency dependence of the calculated neutron scattering spectra between full and minimal hydration states at temperatures between 100 and 300 K. The protein boson peak is observed in the frequency range 1-4 meV at 100 K in both states. The peak frequency in the minimal hydration state shifts to lower than that in the full hydration state. Protein motions with a frequency higher than 4 meV were shown to undergo almost harmonic motion in both states at all temperatures simulated, whereas those with a frequency lower than 1 meV dominate the total fluctuations above 220 K and contribute to the origin of the glass-like transition. At 300 K, the boson peak becomes buried in the quasielastic contributions in the full hydration state but is still observed in the minimal hydration state. The boson peak is observed when protein dynamics are trapped within a local minimum of its energy surface. Protein motions, which contribute to the boson peak, are distributed throughout the whole protein. The fine structure of the dynamics structure factor is expected to be detected by the experiment if a high resolution instrument (<∼20 μeV) is developed in the near future.     

Intramolecular low-frequency vibrations in lysozyme by neutron time-of-flight spectroscopy

High-resolution neutron time-of-flight (TOF) spectra were measured for partly deuterated hen egg-white lysozyme in solution with and without N-acetyl-glucosamine inhibitor bound to the cleft, and in polycrystalline form. Weak but reproducible bands occur at frequencies between 25 and 375 cm^?1. The bands were tentatively assigned on the basis of previous results for homopolypeptides. At very small energy transfers (between about 1 cm^?1 and 40 cm^?1), the TOF spectra show a dependence both on inhibitor binding and crystalline environment. This is interpreted in terms of conformational flexibility.     

Vibrational mode analysis of guanine by neutron inelastic scattering

The low-temperature neutron inelastic spectrum of guanine has been measured. In order to assign the intense peaks observed in this spectrum, a normal mode analysis has been performed, using the Wilson GF-method. The theoretical treatment is based on a non-redundant set of internal coordinates, and a simplified valence force-field approximation. Only the fundamentals have been considered for simulating the internal vibrational mode spectrum. The calculations account for the spectral shape as well as the main observed peaks.Offprint requests to: M. Ghomi     

Comparison of neutron and X-ray scattering of dilute myoglobin solutions.

Experimental results obtained by neutron scattering of dilute solutions of myoglobin are compared with those obtained by X-ray scattering. X-ray scattering remains the more powerful technique at wider angles above 0.3 Å⁻¹, where neutron experiments are less accurate because of low coherent scattering probability and high incoherent background. Neutron scattering is preferable at momentum transfers below 0.2 Å⁻¹; the conditions for applying the contrast variation method for the evaluation of the three basic scattering functions, which are due to shape and internal structure, equation (3), are ideally fulfilled in this region. Furthermore, neutrons allow observation of the hydrogen-deuterium exchange within the protein.     

Hydration structure in natural DNA observed by thermal neutron scattering

Films of highly oriented Na- and LiDNA showing the typical X-ray diffraction patterns for the A-,B-, and C-conformation have been investigated by elastic and quasielastic neutron scattering. Information concerning the question of the DNA-water interaction has been obtained by varying the parameters H₂O/D₂O contrast, humidity, and temperature. Main observations are: A coexistence of one- and three-dimensionally correlated DNA which shifts towards the one-dimensionally correlated C-conformation for high humidity; a coexistence of A-, B-, and C-conformation for NaDNA with a similar humidity dependence; a factor of two increase between the average degree of localization of water hydrogens compared with DNA hydrogens at 75% r.h. for NaDNA; a strong water contribution to layer peaks which are close to the susceptibility maximum of water; a strong temperature dependence of the axial repeat distance for C-DNA; broad quasielastic spectra around the inverse of this distance. The observations are interpreted in terms of a competition between finite three-dimensional correlation and an optimized spatial resonance of nearly one-dimensionally correlated DNA with the correlation of bulk water. The observations are compatible with the concept of water spine formation (Dickerson 1983). The interpretation emphasizes the dynamic character of this mechanism in the region of nearly one-dimensionally correlated DNA.     

Measurement of coherent Debye-Waller factor in in vivo deuterated C-phycocyanin by inelastic neutron scattering.

Quasielastic neutron scattering measurements of dry and 35% D2O hydrated amorphous protein powder of C-phycocyanin were made as a function of temperature ranging from 313K down to 100K. The protein is grown from blue-green algae cultured in D2O and is deuterated up to 99%. The scattering is thus dominated by coherent scattering. Within the best energy resolution of the time-of-flight instrument, which is 28 mueV FWHM, the scattering appears entirely elastic. For this reason we are able to extract a coherent Debye-Waller factor by making an independent measurement of the static structure factor. We observe a considerable difference in the q dependence of the Debye-Waller factor between the dry and hydrated proteins; furthermore, there is an interesting temperature dependence of the Debye-Waller factor that is quite different from that predicted for dense hard-sphere liquids.     

Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen.

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.     

Spectral broadening of the Soret band in myoglobin: an interpretation by the full spectrum of low-frequency modes from a normal modes analysis

In this work the temperature dependence of the Soret band line shape in carbon-monoxy myoglobin is re-analyzed by using both the full correlator approach in the time domain and the frequency domain approach. The new analyses exploit the full density of vibrational states of carbon-monoxy myoglobin available from normal modes analysis, and avoid the artificial division of the entire set of vibrational modes coupled to the Soret transition into "high-frequency" and "low-frequency" subsets; the frequency domain analysis, however, makes use of the so-called short-times approximation, while the time domain one avoids it. Time domain and frequency domain analyses give very similar results, thus supporting the applicability of the short-times approximation to the analysis of hemeprotein spectra; in particular, they clearly indicate the presence of spectral heterogeneity in the Soret band of carbon-monoxy myoglobin. The analyses also show that a temperature dependence of the Gaussian width parameter steeper than the hyperbolic cotangent law predicted by the Einstein harmonic oscillator and/or a temperature dependence of inhomogeneous broadening are not sufficient to obtain quantitative information on the magnitude of an-harmonic contributions to the iron-heme plane motion. However, the dependence of the previous two quantities may be used to obtain semiquantitative information on the overall coupling of the Soret transition to the low-frequency modes and therefore on the dynamic properties of the heme pocket in different states of the protein.     

Biophysical aspects of neutron scattering from vibrational modes of proteins

This review describes a major portion of the published work on neutron scattering experiments aimed at measuring large scale motions in proteins. The importance of these motions for enzyme function and oxygen transport is indicated. The theory applicable to each type of neutron scattering measurement is given and results are discussed with a view to biological relevance. New experiments are suggested and a comparison of neutron scattering data is made with results from other techniques such as raman scattering, infrared absorption, photolysis and molecular dynamics simulations.     

Molecular dynamics of solid-state lysozyme as affected by glycerol and water: a neutron scattering study

Glycerol has been shown to lower the heat denaturation temperature (T(m)) of dehydrated lysozyme while elevating the T(m) of hydrated lysozyme (. J. Pharm. Sci. 84:707-712). Here, we report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions associated with structural relaxation processes were not detected for dehydrated lysozyme in the temperature range of 40 to 450K. Dehydrated lysozyme was found to have the highest T(m) by. Upon the addition of glycerol or water, anharmonicity was recovered above a dynamic transition temperature (T(d)), which may contribute to the reduction of T(m) values for dehydrated lysozyme in the presence of glycerol. The greatest degree of anharmonicity, as well as the lowest T(d), was observed for lysozyme solvated with water. Hydrated lysozyme was also found to have the lowest T(m) by. In the regime above T(d), larger amounts of glycerol lead to a higher rate of change in anharmonic motions as a function of temperature, rendering the material more heat labile. Below T(d), where harmonic motions dominate, the addition of glycerol resulted in a lower amplitude of motions, correlating with a stabilizing effect of glycerol on the protein.     

Equilibrium fluctuations in myoglobin and lysozyme

The angular dependencies of inelastic intensities of Rayleigh scattering of Moessbauer radiation were measured for myoglobin and lysozyme (in the hydration range h = 0.05-0.7). The data were fitted within the framework of model, when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative motions of segments. The best agreement with the experiment at h > 0.05 was obtained when individual motions of small groups together with the cooperative motions of alpha-helices and beta-sheets for lysozyme, and alpha-helices for myoglobin were considered. At further hydration (h = 0.45), mean-square displacements (x2) of both types of motions strongly increase with the increase in hydration degree, while the motions with a large correlation radius (not less than macromolecule radius) remain nearly the same as for h = 0.05. The results of the study of the radial distribution function deduced by Fourier-transform from the diffuse x-ray measurements together with RSMR data allow one to conclude that the water during protein hydration competes with the intramolecular hydrogen bonds, loosens the protein and increases the internal dynamics. Concurrently, water arranges the ordering of macromolecule, which takes the native structure at h = 0.4-0.7. The analysis of auto and cross-correlation functions of bending fluctuations of alpha-helices in the large domain of lysozyme performed by molecular dynamics allows one to come to the final conclusion that it is the difference in the structural organization of myoglobin and lysozyme and not the presence of SS-bonds in lysozyme macromolecule that is responsible for different structural fluctuations in these proteins.     

Low-frequency vibrational modes in proteins: a neutron scattering investigation

The low-frequency dynamics of plastocyanin, an electron transfer copper protein, has been investigated by incoherent neutron scattering at different temperatures. The contribution to the dynamic structure factor arising from H/D exchangeable and non-exchangeable protein protons has been evaluated by analyzing two differently exchanged protein samples. The dynamic structure factor of a hydrated plastocyanin sample with all the exchangeable hydrogens (about 150) replaced by deuterium exhibits an excess of vibrational modes, at about 3.5 meV, reminiscent of the boson peak found in other proteins and glassy systems. When only fast exchangeable hydrogens (about 50) are substituted by deuterium, the protein, besides the above-mentioned peak, shows an additional peak at about 1 meV. These vibrational peaks are discussed in connection with the topological disorder of the systems and the fluctuations of the intramolecular hydrogen bonds.     

Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions)

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.     

Temperature dependence of dynamics of hydrated myoglobin. Comparison of force field calculations with neutron scattering data

Molecular dynamics is used to probe the atomic motions of the carboxy-myoglobin protein as a function of temperature. Simulations of 150 picoseconds in length are carried out on the protein at 20, 60, 100, 180, 220, 240, 260, 280, 300, 320 and 340 K. The simulations attempt to mimic neutron scattering experiments very closely by including a partial hydration shell around the protein. Theoretical elastic, quasielastic and inelastic neutron scattering data are derived from the trajectories and directly compared with experiment. Compared to experiment, the simulation-derived elastic scattering curves show a decrease in intensity as a function of the scattering wavevector, q2. The inelastic and quasielastic spectra show that the inelastic peak is shifted to lower frequency than the experimental value, while quasielastic behavior is in good agreement with experiment. This suggests that the theoretical model is too flexible in the harmonic limit (low temperature), but accurately reproduces high-temperature behavior. Time correlation functions of the intermediate scattering function are determined. At low temperature there is one fast decay process, and at high temperatures there is an additional slow relaxation process that is due to quasielastic scattering. The average atomic fluctuations show that the protein behaves harmonically at low temperatures. At approximately 210 K, a glass-like transition in atomic fluctuations is seen. Above the transition temperature, the atomic fluctuations exhibit both harmonic and anharmonic behavior. Comparison of protein mobility behavior with experiment indicate the fluctuations derived from simulations are larger in the harmonic region. However, the anharmonic region agrees very well with experiment. The anharmonicity is large at all temperatures, with a gradual monotonic increase from 0.5 at 20 K to greater than 0.7 at 340 K without a noticeable change at the glass transition temperature. Heavy-atom dihedral transitions are monitored as a function of temperature. Trends in the type of dihedral transitions that occur with temperature are clearly visible. Dihedral transitions involving backbone atoms occur only above the glass transition temperature. The overall protein behavior results suggest that at low temperatures there is purely vibrational motion with one fast decay process, and above the glass transition temperature there is more anharmonic motion with a fast and a slower relaxation process occurring simultaneously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature-dependent vibrational and conformational dynamics of photosystem II membrane fragments from spinach investigated by elastic and inelastic neutron scattering

Vibrational and conformational protein dynamics of photosystem II (PS II) membrane fragments from spinach were investigated by elastic and inelastic incoherent neutron scattering (EINS and IINS). As to the EINS experiments, the average atomic mean square displacement values of PS II membrane fragments hydrated at a relative humidity of 57% exhibit a dynamical transition at ~230K. In contrast, the dynamical transition was absent at a relative humidity of 44%. These findings are in agreement with previous studies which reported a "freezing" of protein mobility due to dehydration (Pieper et al. (2008) Eur. Biophys. J. 37: 657-663) and its correlation with an inhibition of electron transfer from Q(A)(-) to Q(B) (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). IINS spectra of a sample hydrated at a relative humidity of 57% show a distinct Boson peak at ~7.5meV at 20K, which shifts towards lower energy values upon temperature increase to 250K. This unexpected effect is interpreted in terms of a "softening" of the protein matrix along with the onset of conformational protein dynamics as revealed by the EINS experiments. Information on the density of vibrational states of pigment-protein complexes is important for a realistic calculation of excitation energy transfer kinetics and spectral lineshapes and is often routinely obtained by optical line-narrowing spectroscopy at liquid helium temperature. The data presented here demonstrate that IINS is a valuable experimental tool in determining the density of vibrational states not only at cryogenic, but also at nearly physiological temperatures up to 250K. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.