Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Nanosecond structural dynamics of intrinsically disordered β-casein micelles by neutron spectroscopy

β-casein undergoes a reversible endothermic self-association, forming protein micelles of limited size. In its functional state, a single β-casein monomer is unfolded, which creates a high structural flexibility, which is supposed to play a major role in preventing the precipitation of calcium phosphate particles. We characterize the structural flexibility in terms of nanosecond molecular motions, depending on the temperature by quasielastic neutron scattering. Our major questions are: Does the self-association reduce the chain flexibility? How does the dynamic spectrum of disordered caseins differ from a compactly globular protein? How does the dynamic spectrum of β-casein in solution differ from that of a protein in hydrated powder states? We report on two relaxation processes on a nanosecond and a sub-nanosecond timescale for β-casein in solution. Both processes are analyzed by Brownian oscillator model, by which the spring constant can be defined in the isotropic parabolic potential. The slower process, which is analyzed by neutron spin echo, seems a characteristic feature of the unfolded structure. It requires bulk solvent and is not seen in hydrated protein powders. The faster process, which is analyzed by neutron backscattering, has a smaller amplitude and requires hydration water, which is also observed with folded proteins in the hydrated state. The self-association had no significant influence on internal relaxation, and thus, a β-casein protein monomer flexibility is preserved in the micelle. We derive spring constants of the faster and slower motions of β-caseins in solution and compared them with those of some proteins in various states (folded or hydrated powder).     

Intramembrane Polarity by Electron Spin Echo Spectroscopy of Labeled Lipids

The association of water (D₂O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D₂O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the ¹⁴N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.     

The helix-to-sheet transition of an HIV-1 fusion peptide derivative changes the mechanical properties of lipid bilayer membranes

A short sequence on the gp41 envelope protein of HIV-1 is integral to infection by the virus. Without this sequence, termed the fusion peptide (FP), the virus is far less effective at fusing with the cellular membrane. One of the interesting features of the isolated FP is that it transitions between an α-helical conformation and a β-sheet conformation in lipid bilayer membranes as a function of lipid composition and concentration, and the transition correlates with fusion. To better understand how the conformations of the FP impact lipid bilayer membranes, a variant of the FP that does not strongly promote fusion, termed gp41rk, was studied. Circular dichroism spectroscopy, dynamic light scattering, small-angle neutron scattering (SANS) and neutron spin echo spectroscopy (NSE) were used to relate the conformation of gp41rk to the structure and mechanical properties of lipid bilayer membrane vesicles composed of a 7:3 molar ratio mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol). At a peptide-to-lipid ratio (P/L) of 1/200, it adopts an α-helical conformation, while gp41rk is a β-sheet at a P/L of 1/50 in the unilamellar vesicles. SANS reveals that the lipid bilayer membrane becomes thicker when gp41rk adopts a β-sheet conformation, which indicates that the high-concentration state of the peptide increases the order of the lipid acyl chains. At the same time, NSE demonstrates that the bilayer becomes more rigid, demonstrating that the β-sheet conformation, which correlates with fusion for the native FP sequence, stiffens the bilayer. The results have implications for the function of the FP.     

Particle sizes of purified κ-casein: Metal effect and correspondence with predicted three-dimensional molecular models

κ-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogenous polymers, however, self-associated into nearly spherical uniform particles with an average radius of 8.9 nm as revealed by negatively stained transmission electron micrographs. Evidence is presented that multivalent cations play a role in the stabilization of these spherical particles. Treatment with EDTA causes disruption of theκ-casein particles and leads to a broader size distribution as judged by electron microscopy and dynamic light scattering. The size and shape of the particles are in accord with earlier proposed 3D models forκ-casein that actually predicted participation of divalent cations in the structure.     

Clathrin triskelia show evidence of molecular flexibility

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or “basket”. Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket. We extend the earlier solution studies by performing small-angle neutron scattering (SANS) experiments on isolated triskelia, allowing us to examine a higher q range than that probed by static light scattering. Results of the SANS measurements are consistent with the light scattering measurements, but show a shoulder in the scattering function at intermediate q values (0.016 Å⁻¹), just beyond the Guinier regime. This feature can be accounted for by Brownian dynamics simulations based on flexible bead-spring models of a triskelion, which generate time-averaged scattering functions. Calculated scattering profiles are in good agreement with the experimental SANS profiles when the persistence length of the assumed semiflexible triskelion is close to that previously estimated from the analysis of electron micrographs.     

The structure of lipid bilayers and the effects of general anaesthetics: An X-ray and neutron diffraction study

We have looked for the effects of three clinically used inhalational anaesthetics (nitrous oxide, halothane and cyclopropane) on the structure of lecithin/ cholesterol bilayers. The anaesthetics were delivered to the membranes in the gaseous phase, so that effects at clinical concentrations could be determined.High-resolution X-ray diffraction patterns were recorded out to 4 Å and analyzed using swelling experiments. Parallel neutron diffraction experiments were performed and analyzed using H₂O-²H₂O exchange. Methods were developed which enabled us to obtain confidence limits for the X-ray and neutron structure factors.The resultant X-ray and neutron scattering density profiles clearly define the positions of the principal molecular groups in the unperturbed bilayer. In particular, the high-resolution electron density profiles reveal features directly attributable to the cholesterol molecule. A comparison with the neutron scattering density profiles shows that cholesterol is anchored with its hydroxyl group at the water/hydrocarbon interface, aligned with the fatty acid ester groups of the lecithin molecule. We suggest that this positioning of the cholesterol molecule allows it to act as a thickness buffer for plasma membranes.In the presence of very high concentrations of general anaesthetics, the bilayers show increased disorder while maintaining constant membrane thickness. At surgical concentrations, however, there are no significant changes in bilayer structure at 95% confidence levels. We briefly review the literature previously used to support lipid bilayer hypotheses of general anaesthesia. We conclude that the lipid bilayer per se is not the primary site of action of general anaesthetics.     

Structure and Dynamics of Veiled Virgin Olive Oil: Influence of Production Conditions and Relation to its Antioxidant Capacity

Structure and dynamics of the colloidal dispersions in veiled virgin olive oil (VVOO), the fresh olive juice, were for the first time investigated with different scattering techniques and related to the extraction conditions applied by the olive oil producers. VVOO samples were produced with either the three-phase extraction procedure (oil/externally added water) at different malaxation times, or by the dual-phase extraction procedure (no externally added water). Static light scattering (Small angle light scattering apparatus SALSA), dynamic light scattering, based on a 3D cross-correlation system, a flat cell and a red HeNe-Laser with 632,8 nm wavelength (3D-DLS), classical dynamic light scattering using a goniometer with cylindrical cells and a green laser with 532 nm wavelength, (Green-DLS), and small angle X-ray scattering (SAXS), are the scattering techniques that were used for the analysis of the samples. In addition, samples of VVOO were analyzed with a confocal microscope. SAXS technique gave almost the same results for all the samples of VVOO indicating comparable nano-structure due to the triglyceride backbone. When 3D-DLS and Green DLS were applied to the VVOO samples, quite different results were obtained. In addition, from the microscopic study of the VVOO samples discrete droplets but no anisotropic crystals could be observed. Finally, radical scavenging activity measurements applying Electron Paramagnetic Resonance spectroscopy showed that the antioxidant capacity of the veiled VVO was higher than the one of the filtered oils. Between the two oil extraction systems the dual phase one seems to be more appropriate for the production of stable and rich in minor constituents olive oils.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.     

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.     

Thermal conductivity of ferromagnetic metallic La0.95Ag0.05MnO3 manganites: role of carrier,spin waves and lattice–impurity scattering

The lattice contribution to the thermal conductivity (κ_ph) of La_0.95Ag_0.05MnO₃ manganites is theoretically analysed within the framework of Kubo model. The theory is formulated when thermal conduction is limited by the scattering of phonons from defects, grain boundaries, charge carriers, spin waves and phonons. The lattice thermal conductivity dominates in Ag-doped manganites and is artefact of strong phonon–impurity and phonon–phonon scattering mechanism in the ferromagnetic metallic state. The electronic contribution to the thermal conductivity (κ_e) is estimated following the Wiedemann–Franz law. Another important contribution in the metallic phase should come from spin waves (κ_m). It is noticed that κ_m increases with a T² dependence on the temperature. The behaviour of the thermal conductivity in manganites is determined by competition among the several operating scattering mechanisms for the heat carriers and a balance between electron, magnon and phonon contributions.     

Neutron spin echo studies on ferritin: free-particle diffusion and interacting solutions

The dynamics of proteins are often studied by means of quasielastic neutron scattering (QENS), for example by time-of-flight methods. The spatial dimensions (10-20 nm) present in protein solutions are accessible by neutron scattering. In this article, a systematic study of diffusive dynamics of ferritin and apoferritin (=ferritin without iron core) is presented. Apoferritin consists of a spherical shell built of 24 protein units and carries net negative charge at pH 5. We have studied diffusive dynamics of ferritin solutions by neutron spin echo (NSE). We pay attention to an important feature of this technique compared to other QENS methods, which being the usage of a broad wavelength band. Using a more sophisticated fit function than usually used in NSE, we find as expected in low concentrated systems that the diffusion coefficient approaches the free-particle value of apoferritin and coincides with the diameter of the apoferritin shell (12.2 nm). In interacting solutions, the NSE results reveal that the dynamic picture of this complex liquid is dominated by slowing down of the dynamics. In low-salt solutions, a structure factor peak appears due to ordering of the ferritin molecules on the length scale of several intermolecular distances. We discuss the usage of different NSE fit functions for interacting solutions near the structure factor peak. Comparison of the dependence of elastic and dynamic data on the scattering vector value shows the influence of indirect interactions on the dynamic picture, irrespective of the way of data analysis, which being necessary due to the broad wavelength spectrum.     

Partial area of cholesterol in monounsaturated diacylphosphatidylcholine bilayers

The influence of cholesterol on the structural parameters of phosphatidylcholine bilayers is studied by small-angle neutron scattering on unilamellar liposomes. Monounsaturated diacylphosphatidylcholines diCn:1PC with the length of acyl chains n = 14, 18 and 22 carbons are used. We confirm that the bilayer thickness increases with increasing concentration of cholesterol for all studied diCn:1PCs. However, partial areas per diCn:1PC and cholesterol molecule on lipid–water interface are found not to depend of cholesterol concentration. The partial area per cholesterol molecule is 0.24 nm². In addition, the partial area per diC18:1PC is larger than that for diC14:1PC and diC22:1PC.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

The structure of detergent-resistant membrane vesicles from rat brain cells

The size and the bilayer thickness of detergent-resistant membranes isolated from rat brain neuronal membranes using Triton X-100 or Brij 96 in buffers with or without the cations, K⁺/Mg²⁺ at a temperature of either 4 °C or 37 °C were determined by dynamic light scattering and small-angle neutron scattering. Regardless of the precise conditions used, isolated membrane preparations consisted of vesicles of ∼ 100 to 200 nm diameter as determined by dynamic light scattering methods, equating to an area of the lipid based membrane microdomain size of 200 to 400 nm diameter. By means of small angle neutron scattering it was established that the average thickness of the bilayers of the complete population of detergent-resistant membranes was similar to that of the parental membrane at between 4.6 and 5.0 nm. Detergent-resistant membranes prepared using buffers containing K⁺/Mg²⁺ uniquely formed unilamellar vesicles while membranes prepared in the absence of K⁺/Mg²⁺ formed a mixture of uni- and oligolamellar structures indicating that the arrangement of the membrane differs from that observed in the presence of cations. Furthermore, the detergent-resistant membranes prepared at 37 °C were slightly thicker than those prepared at 4 °C, consistent with the presence of a greater proportion of lipids with longer, more saturated fatty acid chains associated with the Lo (liquid-ordered) phase. It was concluded that the preparation of detergent-resistant membranes at 37 °C using buffer containing cations abundant in the cytoplasm might more accurately reflect the composition of lipid rafts present in the plasma membrane under physiological conditions.     

Contrast variation studies of clathrin coated vesicles by small-angle neutron scattering

Structural information on clathrin coated vesicles has been obtained by small angle neutron scattering using contrast variation. A characteristic peak in the neutron scattering profile, which is apparent in 75 % D₂O, as well as in H₂O, disappears when contrast matching the protein component of the coated vesicles in 42% D₂O. Neutron, as well as dynamic, light scattering give a coated vesicle size of about 900 Å in H₂O and D₂O, but for neutron scattering the diameter decreases when matching out the protein coat of the clathrin coated vesicles. From the match point for the clathrin coated vesicles it is demonstrated that the clathrin cages do contain internal membrane. The mass of 34 MD and composition of 75% protein and 25% lipid found from the analysis of the small-angle scattering data are both in good agreement with the values reported in the literature. Electron microscopy gives an average outer diameter of 880 Å for the coated vesicles and an average diameter of 460 Å for the vesicle itself. Offprint requests to: Correspondence to: R. Bauer     

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.     

SANS investigation of the photosynthetic machinery of Chloroflexus aurantiacus

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.     

Using polarization analysis to separate the coherent and incoherent scattering from protein samples

Polarization analysis was used to separate experimentally the coherent and spin-incoherent nuclear static scattering functions, from a representative set of samples of interest for protein studies. This method had so far limited application in the study of amorphous materials, despite the relevance of the information that it provides. It allows, for instance, the experimental determination of the structure factor of materials containing a significant amount of hydrogen atoms, avoiding the contamination of measurements by a non-negligible incoherent background. Knowledge of the relative importance of the coherent and incoherent terms at different Q-values is also a pre-requisite for the interpretation of quasielastic neutron scattering experiments, performed at instruments in which the total dynamic scattering function is measured, such as conventional time-of-flight and backscattering spectrometers. Combining data from different instruments, it was possible to cover a wide Q-range, from the small-angle region (0.006 < Q < 0.04 Å^− 1) to the wide-angle region (up to ≈ 2.35 Å^− 1). Quantitative information was obtained on the fraction of coherent to spin-incoherent scattering from different protein samples: deuterated and protonated protein powders at different hydration levels and solutions of protonated proteins in D₂O at different concentrations. The results obtained are discussed in the context of the validity of the assumptions generally made when interpreting quasielastic neutron scattering experiments performed without polarization analysis.     

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.