Neutron scattering measurements of intact cells show changes after heat shock consistent with an increase in molecular crowding

Molecular crowding has been shown to be important in many cellular processes. The crowded environment in the cell results in a significant proportion of the cellular water being in contact with macromolecules such as proteins and DNA. These interfacial water molecules show a reduced dynamic motion that has been observed with isolated macromolecules using several biophysical techniques. Previously we investigated the inelastic neutron scattering properties of water closely associated with isolated biomolecules, and showed that interfacial water is strongly perturbed, as judged by its energy transfer spectrum. Here we have probed living cells using inelastic and quasielastic neutron scattering. We have found that mild heat stress ('heat shock'), which causes some proteins to become unfolded in the cell, results in changes in the inelastic neutron scattering in the librational region (45-130 meV). Heat shock also causes a narrowing of the quasielastic scattering peak. These changes can be understood in terms of an increase in the proportion of interfacial water molecules, and a net reduction in proton dynamics.     

Metallodrug-protein interaction probed by synchrotron terahertz and neutron scattering spectroscopy

This experimental work applied coherent synchrotron-radiation terahertz spectroscopy and inelastic neutron scattering to address two processes directly associated with the mode of action of metal-based anticancer agents that can severely undermine chemotherapeutic treatment: drug binding to human serum albumin, occurring during intravenous drug transport, and intracellular coordination to thiol-containing biomolecules (such as metallothioneins) associated with acquired drug resistance. Cisplatin and two dinuclear platinum (Pt)- and palladium (Pd)-polyamine agents developed by this research group, which have yielded promising results toward some types of human cancers, were investigated. Complementary synchrotron-radiation-terahertz and inelastic neutron scattering data revealed protein metalation, through S- and N-donor ligands from cysteine, methionine, and histidine residues. A clear impact of the Pt and Pd agents was evidenced, drug binding to albumin and metallothionein having been responsible for significant changes in the overall protein conformation, as well as for an increased flexibility and possible aggregation.     

Orientational and rotational velocity correlation functions for water-hydrating B- and Z-DNA double helices

The molecular dynamics simulations reported earlier for the structure and dynamics of water molecules hydrating B- and Z-DNA double helices are analyzed for the orientational correlation functions and the proton rotational velocity autocorrelation functions. The spectra of the rotational velocity autocorrelation functions obtained from the simulation results are compared with the neutron inelastic scattering experiments on hydrated Na-DNA samples. The results predict a small frequency component associated with water molecules bound to the double helices that disappears for waters away from the double helix.     

Vibrational modes of hemoglobin in red blood cells.

Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective.     

Low-temperature molecular dynamics simulations of horse heart cytochrome c and comparison with inelastic neutron scattering data

Molecular dynamics (MD) simulation combined with inelastic neutron scattering can provide information about the thermal dynamics of proteins, especially the low-frequency vibrational modes responsible for large movement of some parts of protein molecules. We performed several 30-ns MD simulations of cytochrome c (Cyt c) in a water box for temperatures ranging from 110 to 300 K and compared the results with those from experimental inelastic neutron scattering. The low-frequency vibrational modes were obtained via dynamic structure factors, S(Q, ω), obtained both from inelastic neutron scattering experiments and calculated from MD simulations for Cyt c in the same range of temperatures. The well known thermal transition in structural movements of Cyt c is clearly seen in MD simulations; it is, however, confined to unstructured fragments of loops Ω₁ and Ω₂; movement of structured loop Ω₃ and both helical ends of the protein is resistant to thermal disturbance. Calculated and experimental S(Q, ω) plots are in qualitative agreement for low temperatures whereas above 200 K a boson peak vanishes from the calculated plots. This may be a result of loss of crystal structure by the protein–water system compared with the protein crystal.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

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.     

Inelastic neutron scattering analysis of picosecond internal protein dynamics. Comparison of harmonic theory with experiment

The experimental inelastic neutron scattering spectrum of a protein, the bovine pancreatic trypsin inhibitor (BPTI), in a powder sample is presented together with the generalized density of states, G(omega), as a function of the frequency, omega, derived from the scattering data. The experimental results are compared with calculations from two different normal mode analyses of BPTI. One of these, based on an improved model, gives a calculated spectrum and density of states in general agreement with those obtained experimentally; the other, based on an earlier model, shows considerable disagreement. The important improvements in the newer normal mode analysis are the explicit treatment of all atoms (non-polar as well as polar hydrogens are included) and a modified truncation scheme for the long-range electrostatic interactions. The fact that the inelastic neutron scattering measurements can distinguish between the two theoretical models makes clear their utility for the analysis of protein dynamics.     

Temperature dependence of the low frequency dynamics of myoglobin. Measurement of the vibrational frequency distribution by inelastic neutron scattering.

Inelastic neutron scattering spectra of myoglobin hydrated to 0.33 g water (D2O)/g protein have been measured in the low frequency range (1-150 cm-1) at various temperatures between 100 and 350 K. The spectra at low temperatures show a well-resolved maximum in the incoherent dynamic structure factor Sinc(q, omega) at approximately 25 cm-1 and no elastic broadening. This maximum becomes gradually less distinct above 180 K due to the increasing amplitude of quasielastic scattering which extends out to 30 cm-1. The vibrational frequency distribution derived independently at 100 and 180 K are very similar, suggesting harmonic behavior at these temperatures. This result has been used to separate the vibrational motion from the quasielastic motion at temperatures above 180 K. The form of the density of states of myoglobin is discussed in relation to that of other amorphous systems, to theoretical calculations of low frequency modes in proteins, and to previous observations by electron-spin relaxation of fractal-like spectral properties of proteins. The onset of quasielastic scattering above 180 K is indicative of a dynamic transition of the system and correlates with an anomalous increase in the atomic mean-squared displacements observed by M?ssbauer spectroscopy (Parak, F., E. W. Knapp, and D. Kucheida. 1982. J. Mol. Biol. 161: 177-194.) and inelastic neutron scattering (Doster, W., S. Cusack, and W. Petry, 1989. Nature [Lond.]. 337: 754-756.) Similar behavior is observed for a hydrated powder of lysozyme suggesting that the low frequency dynamics of globular proteins have common features.     

Low-frequency modes of peptides and globular proteins in solution observed by ultrafast OHD-RIKES spectroscopy

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.     

Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the “boson peak” observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure. Received: 12 October 1998 / Revised version: 19 February 1999 / Accepted: 1 March 1999     

Relationship between structure, dynamics and function of hydrated purple membrane investigated by neutron scattering and dielectric spectroscopy

We investigated the influence of hydration water on the relationship between structure, dynamics and function in a biological membrane system. For the example of the purple membrane (PM) with its protein bacteriorhodopsin (BR), a light-driven proton pump, complementary information from neutron diffraction, quasi-elastic neutron scattering (QENS) and dielectric spectroscopy will form a comprehensive picture of the structural and dynamic behavior of the PM in the temperature range between 150 and 290 K. Temperature- and humidity-dependent changes in the membrane system influence the accessibility of the different photocycle intermediates of BR. The melting of the 'freezing bound water' between 220 and 250 K could be related to the transition from the M1 to the M2 intermediate, which represents the key step in the photocycle. The dynamic transition in the vicinity of 180 K was shown to be necessary to ensure that the M1 intermediate can be populated and that the melting of crystallized bulk water above 255 K enables the completion of the photocycle.     

Temperature dependence of the generalized frequency distribution of water molecules: comparison of experiments and molecular dynamics simulations

The results of inelastic neutron scattering experiments on water in the temperature interval 300–623 K along the coexistence curve are compared with data obtained from molecular dynamics simulations. In general, a good agreement between experiments and calculations is observed and it serves as a satisfactory test of the potential models employed. The temperature dependence of the generalized frequency distribution of water molecules obtained by both experiment and computer simulation demonstrates the accordance with the temperature evolution of the water structure, extracted from neutron and X-ray diffraction measurements.     

Frozen spin targets in ribosomal structure research.

Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation. A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction. Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins. Pure deuteron spin labels and proton spin labels are created by NMR saturation. We report on results obtained from the large subunit of E. coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN. The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation. Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K. At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target). Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively. The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature. This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs.     

Normal vibrations of -glycylglycine

A complete normal coordinate analysis, infrared absorption, and inelastic neutron scattering studies on α-glycylglycine are reported. A Urey-Bradley force field which is also valid for N-deuterated sample is obtained. Assignments in the low-frequency region are based on inelastic neutron spectrum obtained in one phonon and cubic approximation.     

Direct Measurement of Hydration-Related Dynamic Changes in Lysozyme using Inelastic Neutron Scattering Spectroscopy

Abstract

Inelastic neutron scattering spectroscopy is used to investigate dynamic changes in lysozyme powder at two different low D,0 hydrations (0.07g D²,O/g protein and 0,20 g D²,O/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 molccule[M. Levitt, C. Sanderand P. S. Stern, J. Mol. Biol. 181. 423 (1985). B.Brooks and M.Karplus.Prot. 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.     

Protein interactions and dynamics probed by quantum chemistry, computer simulations and neutron experiments

We review some recent experiments and calculations on aspects of the structure and dynamics of proteins and related systems. The use of quantum chemical techniques to determine geometries and energies of supramolecular complexes of biological interest is illustrated, and the concomitant development of empirical energy functions for use in protein simulations outlined. We describe how simulations of crystalline peptides and amino-acids using an empirical force field can be combined with appropriate coherent and incoherent inelastic neutron scattering experiments to elucidate the characteristics of lattice vibrations and diffusive atomic motions in the crystals. The application of molecular dynamics simulations to the interpretation of incoherent neutron scattering experiments on proteins is examined and the resulting ideas on the general characteristics of protein motion discussed in terms of their functional implications.     

Possibility of wide-angle neutron scattering in the study of proteins and nucleic acids in solutions

A method is proposed for calculating wide-angle neutron scattering curves of biopolymers at any fraction of heavy water (D2O) in solution. The method permits to accurately take into account the phenomenon of deuteroexchange. By this method neutron scattering curves of proteins and DNA have been calculated. The calculations have shown that at optimal fractions of D2O in solution the profiles of neutron scattering curves and their sensitivity to conformational rearrangements in protein molecules turned out to differ very little from those of corresponding X-ray curves. Thus the neutron scattering curves do not contain any additional information (as compared with those contained in X-ray scattering curves) on the structure of proteins in solution. On the contrary, neutron and X-ray scattering curves of DNA differ significantly at all fractions of D2O in solution and therefore the methods of wide-angle neutron and X-ray scattering could become mutually complementary in studying the structure of nucleic acids in solution.     

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)