Temperature dependence of dielectric relaxations in alpha-elastin coacervate: evidence for a peptide librational mode

The dielectric permittivity of alpha-elastin coacervate is reported over the frequency range of 1 MHz to 1000 MHz and the temperature dependence from 6.8 degrees C to 70 degrees C is also reported. A temperature-dependent simple Debye-type relaxation is observed with a correlation time of 8 nsec (40 degrees C) which is similar to that of the polypentapeptide of elastin (i.e. 7 nsec at 40 degrees C) where the band has been assigned to a peptide librational mode. By analogy this allows for the first assignment of a peptide librational mode in a naturally occurring polypeptide or protein. The strong spectrally localized band indicates a regularity of structure. The low temperature dependence of the correlation time, giving a 1.7 kcal/mole enthalpy of activation, is consistent with torsional motions associated with a peptide librational mode.     

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein. Secondary structure motion in a 150 picosecond trajectory

A 150 picosecond molecular dynamics computer simulation of the C-terminal fragment of the L7/L12 ribosomal protein from Escherichia coli is reported. The molecular dynamics results are compared with the available high-resolution X-ray data in terms of atomic positions, distances and positional fluctuations. Good agreement is found between the molecular dynamics results and the X-ray data. The form and parameters of the interaction potential energy function and the procedures for deriving it are discussed. Some current misunderstandings concerning the ways of evaluating the efficiency of molecular dynamics algorithms and of application of bond-length constraints in protein simulations are cleared up. The 150 picosecond trajectory has been scanned in a search for correlated motions within and between secondary structure elements. The beta-strands have diffusional stretching modes, and uncorrelated transversal displacements. The dynamic analysis of alpha-helices shows a variety of features. The atomic fluctuations differ between the helix ends; this effect reflects long time-scale motions. Two alpha-helices, alpha A and alpha C, show diffusive longitudinal stretching modes. The third helix, alpha B, has a correlated asymmetric longitudinal stretching; the N-terminal part dominates this behaviour. Furthermore, alpha B presents a librational motion with respect to the other parts of the molecule with a frequency of approximately 5 cm-1. This motion is coupled to helix stretching. Interestingly, the regions of highly conserved residues contain the most mobile parts of the molecule.     

An ESR Spin label study of structural and dynamical properties of oriented lecithin-cholesterol multibilayers.

Oriented dipalmitoyllecithin-cholesterol multibilayers with 11% water have been studied with the cholestane spin label. From the ESR spectra the order parameters and the mobility of the spin label about its long axis have been calculated. The results on pure lecithin multibilayers indicate a transition from gel to liquid crystalline phase at 52 plus or minus 2 degrees C. In the gel phase the lecithin alkyl chains are highly ordered, but tilted with respect to the normal to the bilayers by about 25 degrees. Above 52 degrees C the tilt disappears and the mobility of the cholestane spin label increases, indicating an increase of mobility of the lecithin alkyl chains. When cholesterol is added, below about 52 degrees C a decrease of order is found. Furthermore, already small cholesterol contents (smaller than or equal to 10 mole %) remove the tilt. Above about 52 degrees C cholesterol improves the order by decreasing the amplitude of the librational motions. Cholesterol lowers the transition temperature of the system and reduces the mobility of the lecithin alkyl chains in the liquid crystalline phase. However an increase in mobility is found at cholesterol contents up to 10 mole %. A very broad phase transition is observed at 50 mole % cholesterol. In all systems an increase in temperature results in a reduction of order through an increase of the amplitude of the librational motions of the molecules. The librational motions are to some extent cooperative. The asymmetry of the order matrix is found to be a measure for the lateral ordering. Cholesterol increases the lateral ordering, indicating that the flat cholesterol molecules orient parallel to each other.     

The motion of aromatic molecules in bile acid micelles

Deuterium spin-lattice relaxation time (T₁) of perdeuterobenzene and perdeuteronaphthalene dissolved in aqueous solutions of sodium deoxycholate and sodium cholate was studied. By comparing the experimental data and theoretical calculations, it was concluded that benzene and naphthalene are not held stationary in the bile acid micelles, and their molecular planes have certain librational motion. In addition, benzene undergoes rapid rotation about the C₆ axis, but naphthalene does not rotate in its molecular plane.     

Librational motion of an "immobilized" spin label: hemoglobin spin labeled by a maleimide derivative.

The spin label Tempo-maleimide, when "immobilized" in hemoglobin, is shown to exhibit motional fluctuation whose amplitude and/or frequency depend on temperature and solution conditions. These motional fluctuations are observable by several electron spin resonance techniques. For desalted hemoglobin the fluctuations are detectable at approximately -15 degrees C using saturation transfer techniques and at approximately +25 degrees C using line-width measurements of normal absorption spectra. In ammonium sulfate precipitated hemoglobin, however, motional fluctuations are not detectable by either technique up to at least 40 degrees C. The most probable mechanism for spin-label motion appears to be either fluctuations in protein conformation which affect the label binding site or conformational transitions of the nitroxide ring itself. These motional fluctuations are shown to introduce a librational character to the overall label motion during hemoglobin rotational diffusion, with the librational motion significantly affecting the use of spin-label spectral shapes to calculate hemoglobin rotational correlation times.     

13C and 15N NMR and time-resolved fluorescence depolarization study of bovine carbonic anhydrase--4-methylbenzenesulfonamide complex

The influence of the binding of the high-affinity inhibitor, 4-methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N- and 13C-NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time-resolved fluorescence depolarization measurements. Our experiment provides evidence that the stoichiometry of the interaction of 4-methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N-NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme-inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.     

Glycerolipids: common features of molecular motion in bilayers

In the present study, analysis of 2H NMR line-shape and spin-lattice relaxation behavior has been used to investigate the dynamics of several glycolipid and phospholipid bilayers. The gel-phase spectra of these lipids labeled at the C3 position of the glycerol backbone are broad (approximately 90 kHz) and characteristic of fast-limit axially asymmetric motion. Moreover, anisotropic spin-lattice relaxation is observed in all of these systems. The line-shape and relaxation features of the lipids in the gel phase were best simulated by using a fast-limit three-site jump model, with relative site populations of 0.46, 0.34, and 0.20. This motion is associated with an internal jump about the C2-C3 bond of the glycerol backbone. A second motion, rotation about the long axis of the molecule, is needed to account for the observed temperature dependence of the quadrupolar echo amplitude and the spectral line shape above and below the gel to liquid-crystalline phase transition temperature. On the other hand, the gel-phase spectra of phospholipids labeled at the C2 position of the glycerol backbone are also characterized by a fast internal motion, which is simulated by a two-site librational jump. The results indicate that the glycerol backbone dynamics of the glycolipid and phospholipid systems investigated in this study can be described in terms of common fast internal motions and a slower whole molecule axial motion. These results are compared with previous dynamic studies of similar systems.     

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide

Summary The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 s at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.     

Bound ligand motion in crystalline carboxypeptidase A.

Deuterium NMR spectra for the phenyl ring deuterons have been obtained for D-phenylalanine, L-phenylalanine, phenylacetic acid, and phenyl propionic acid in randomly oriented crystals of carboxypeptidase A as a function of water content. The spectra are analyzed using a two-site jump model for phenyl ring pi-flips when the ligand is bound to the protein, and the model includes the possibility that the ligand may exchange with isotropic or unbound environments within the crystal. Although the binding pocket may impose local dynamical constraints, a complete pi-flip motion is consistent with the spectra of all ligands at all water contents. The rate constants for the pi-flip at 298 K are found to be 7.5 x 10(5) S-1, 1.9 x 10(6) S-1, 4.0 x 10(6) S-1, and 4.0 x 10(6) S-1 for L-phenylalanine, D-phenylalanine, phenyl propionic acid, and phenylacetic acid, respectively, at water activity of 0.98. The pi-flip rate for the ligand bound to the enzyme increases with water content. Assuming that the activation barrier may be written, delta G+2 = delta G+2o + baw, where aw is the water activity, and the value of b is -1.9 kcal/mol for phenylacetic acid and phenyl propionic acid, -1.3 kcal/mol for L-phenylalanine, and -2.1 kcal/mol for D-phenylalanine. Phenylacetic acid crystals were studied as an example of a phenyl ring motion that is highly constrained by a known and symmetrical packing environment. The deuterium spectra are complex and are not consistent with pi-flip motions, but they are consistent with a superposition of ring jump motions of 24 degrees, 34 degrees, and 72 degrees, with probabilities in the ratio of 1:1:2. Because of the limited space for motion imposed by the tight packing in the crystal, these motions must be highly cooperative and probably locally coherent; however, the spectra by themselves do not prove this intuitively reasonable hypothesis.     

Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.     

Probe dependence of correlation times in heme-free extrinsically labeled human hemoglobin

Human heme-free hemoglobin was labeled at beta 93 with either N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate (AEDANS) or fluorescein iodoacetamide (FIA), at beta 1 with pyridoxal-5-phosphate (PLP) and at the heme pocket with anilinonaphthalene-8-sulfonate (ANS). The correlation times associated with these probes ranged from approximately 12 ns for FIA and AEDANS to nearly 20 ns for ANS and PLP. This indicates the presence of internal flexibility in apohemoglobin with librational motions dominated by the mobilities of the monomeric subunits and of the entire dimeric molecules, variously weighted by the different probes. It was not possible to detect motions characterized by correlation times of about 5 ns such as were present in AEDANS-labeled oxyhemoglobin.     

An alternative derivation of the equations of motion in torsion space for a branched linear chain.

Torsion space molecular dynamics may be more efficiently encoded if the global motions are separated from the internal motions. The equations of motion for single, non-cyclic chains are shown to be first order in the backbone angle parameters when the global frame of reference is ignored and second order otherwise. Adding a simple heuristic substitute for the global motions enables the encoding of dynamics for mixed constrained/unconstrained model systems.     

Activation of nanoscale allosteric protein domain motion revealed by neutron spin echo spectroscopy

NHERF1 is a multidomain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length-scales and on submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE.     

Impact of enzyme motion on activity

Experimental and theoretical data imply that enzyme motion plays an important role in enzymatic reactions. Enzyme motion can influence both the activation free energy barrier and the degree of barrier recrossing. A hybrid theoretical approach has been developed for the investigation of the relation between enzyme motion and activity. This approach includes both electronic and nuclear quantum effects. It distinguishes between thermally averaged promoting motions that influence the activation free energy barrier and dynamical motions that influence the barrier recrossings. Applications to hydride transfer in liver alcohol dehydrogenase and dihydrofolate reductase resulted in the identification and characterization of important enzyme motions. These applications have also led to the proposal of a network of coupled promoting motions in enzymatic reactions. These concepts have important implications for protein engineering and drug design.     

Applicability of hydrodynamic analyses of spermatozoan motion.

This paper investigates the applicability of a simple hydrodynamics theory due to Gray & Hancock (1955), and extended by Brokaw (1970), to the analysis of the motions of sea urchin, rabbit, and bull spermatozoa. Experimental procedures for filming and analysing the swimming motions are presented. A detailed discussion of the agreement between the experimental and theoretical results is given, and the limitations of the theory in analysing the motion of mammalian spermatozoa are discussed.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

Covariation of backbone motion throughout a small protein domain

The synchronization (correlation) of conformational fluctuations in folded proteins may influence the rates of enzyme catalysis and ligand binding as well as the stabilities of native proteins and their complexes. However, experimental detection of correlated motions remains difficult. Herein, we present an analysis of the covariation of NMR-derived backbone dynamical parameters among a family of ten mutants of a small protein. Both the spatial restriction and the time scales of backbone motions exhibit a higher degree of covariation than would be expected if the internal motions of each group were independent, providing experimental support for correlated dynamics. Application of this approach to other proteins may reveal dynamical correlations that influence catalysis, ligand-binding and/or protein stability.     

13C Nuclear magnetic relaxation study of segmental dynamics of hyaluronan in aqueous solutions

(13)C spin-lattice relaxation times (T(1)) and nuclear Overhauser enhancements (NOE) were measured as a function of temperature and magnetic field strength for the hetero-polysaccharide hyaluronan in water solutions. The relaxation data of the endocyclic ring carbons were successfully interpreted in terms of chain segmental motions by using the bimodal time-correlation function of Dejean de la Batie, Laupretre and Monnerie. On the basis of the calculated correlation times for segmental motion and amplitudes of librational motions of the C-H vectors at the various carbon sites of the HA repeating unit, we concluded that intramolecular hydrogen bonding of the secondary structure of HA plays a major role in the conformational flexibility of this carbohydrate molecule. The internal rotation of the free hydroxymethyl groups about the exocyclic C-5-C-6 bonds superimposed on segmental motion has been described as a diffusion process of restricted amplitude. The rate and amplitude of the internal rotation indicate that the hydroxymethyl groups are not involved in intramolecular hydrogen bonding. Finally, the motional parameters describing the local dynamics of the HA chain were correlated with the secondary structure of HA in aqueous solutions.