Sampling of Protein Dynamics in Nanosecond Time Scale by 15N NMR Relaxation and Self-Diffusion Measurements

Abstract

This paper presents a procedure for detection of intermediate nanosecond internal dynamics in globular proteins. The procedure uses ¹H-¹⁵N relaxation measurements at several spectrometer frequencies and hydrodynamic calculations based on experimental self-diffusion coefficients. New heteronuclear experiments, using pulse field gradients, are introduced for the measurement of translation diffusion coefficients of ¹⁵N labeled proteins. An advanced interpretation of recently published (Luginbühl et al., Biochemistry, 36, 7305–7312 (1997)) backbone amide ¹⁵N relaxation data, measured at two spectrometers (400 and 750 MHz for ¹H) for N-terminal DNA-binding domain (1–63) of 434 repressor, is presented. Non-applicability of commonly used fast (picosecond) dynamics model (FD) was justified by (i) poor fit of relaxation data by the FD model-free spectral density function both for isotropic and anisotropic models of the overall molecular tumbling; (ii) specific dependence of the overall rotation correlation times calculated from T₁/T₂ ratio on the spectrometer frequency; (iii) mismatch of the ratio of longitudinal ¹⁵N relaxation times T₁, measured at different spectrometer frequencies, in comparison with that anticipated for the IT) model; (iv) significantly underestimated overall rotation correlation time provided by the FD model (5.50±0.15 and 5.80±0.15 ns for 750 and 400 MHz spectrometer frequency respectively) in comparison with correlation time obtained from hydrodynamics. On the other hand, all relaxation and hydrodynamics data are in good correspondence with the model of intermediate (nanoseconds) dynamics. Overall rotation correlation time of 7.5±0.7 ns was calculated from experimental translation self-diffusion rate using hydrodynamics formalism (Garcia de la Torre, J. and Bloomfield, V.A. Quart. Rev. Biophys., 14, 81–139 (1981)). The statistical analysis of ¹⁵N relaxation data along with the hydrodynamic consideration clearly revealed that most of the residues in 434(1–63) repressor are involved in the nanosecond internal dynamics characterized by the the mean order parameters of 0.59±0.06 and the correlation times of ca. 5 ns.     

Amide Proton Spin-Lattice Relaxation in Polypeptides: A Field-Dependence Study of the Proton and Nitrogen Dipolar Interactions in Alumichrome

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the ¹H spinlattice relaxation times (T₁'s) are distinct in the Al³⁺-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of ¹H—¹H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T₁^-1 and Σr^-6 at the three magnetic fields. Considering the very sensitive r-dependence of T₁, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T₁^-1 is proposed to mainly originate from the ¹H-¹⁴N dipolar interaction: this was supported by comparison with measurements on an ¹⁵N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {¹H}—¹H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the ¹H—¹H dipolar mechanism to cause NOE's. From the slope and intersect values of T₁^-1 vs. Σr^-6 linear plots, a number of independent estimates of τ_r, the rotational correlation time, were derived. These and the field-dependence of the T₁'s yield a best estimate <τ_r> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τ_r> [unk] 0.41 ns, previously determined from ¹³C and ¹⁵N spin-lattice relaxation data.     

Proton Nuclear Magnetic Resonance Relaxation Measurements in Frog Muscle

Proton nuclear magnetic resonance (NMR) relaxation measurements are reported for frog muscle as a function of temperature and Larmor frequency. Each T_1ρ, T₂, and T₁ measurement covered a time domain sufficient to identify the average relaxation time for most intracellular water. Using regression analysis the data were fit with a model where intracellular water molecules are exchanging between a large compartment in which mobility is similar to ordinary water and a small compartment in which motion is restricted. The regression results suggest that: the restricted compartment exhibits a distribution of motions skewed toward that of free water; the residence time of water molecules in the restricted compartment is approximately 1 ms; and, the activation entropy for some water molecules in the restricted compartment is negative.     

Growth-induced Water Potentials in Plant Cells and Tissues

A physical analysis of water movement through elongating soybean (Glycine max L. Merr.) hypocotyls was made to determine why significant water potentials persist in growing tissues even though the external water potentials were zero and transpiration is virtually zero. The analysis was based on a water transport theory modified for growth and assumed that water for growing cells would move through and along the cells in proportion to the conductivity of the various pathways.

Water potentials calculated for individual cells were nearly in local equilibrium with the water potentials of the immediate cell surroundings during growth. However, water potentials calculated for growing tissue were 1.2 to 3.3 bars below the water potential of the vascular supply in those cells farthest from the xylem. Only cells closest to the xylem had water potentials close to that of the vascular supply. Gradients in water potential were steepest close to the xylem because all of the growth-sustaining water had to move through this part of the tissue. Average water potentials calculated for the entire growing region were −0.9 to −2.2 bars depending on the tissue diffusivity.

For comparison with the calculations, average water potentials were measured in elongating soybean hypocotyls using isopiestic thermocouple psychrometers for intact and excised tissue. In plants having virtually no transpiration and growing in Vermiculite with a water potential of −0.1 bar, rapidly growing hypocotyl tissue had water potentials of −1.7 to −2.1 bars when intact and −2.5 bars when excised. In mature, nongrowing hypocotyl tissue, average water potentials were −0.4 bar regardless of whether the tissue was intact or excised.

The close correspondence between predicted and measured water potentials in growing tissue indicates that significant gradients in water potential are required to move growth-associated water through and around cells over macroscopic distances. The presence of such gradients during growth indicates that cells must have different cell wall and/or osmotic properties at different positions in the tissue in order for organized growth to occur. The mathematical development used in this study represents the philosophy that would have to be followed for the application of contemporary growth theory when significant tissue water potential gradients are present.

     

The Dielectric Relaxation of Water in Plant Tissue

Thermally stimulated depolarization current (TSDC) measurementson plant leaves and stems of six different species in the temperaturerange of 77–300 K revealed the existence of three differentdispersions. The first dispersion at low temperatures, whichis attributed to the relaxation of loosely bound water moleculeswas studied in detail in an attempt to obtain information onthe possible structures of water in plant tissue. Its characteristicsdiffer for various plant tissues, indicating a different organizationof water in those plant tissues. The dispersion can be describedby a continuous distribution of relaxation times t with boththe activation energy W and the pre-exponential factor To inthe Arrhenius equation being distributed parameters. The spectrumof W and T_o was determined for E. globulus and O. europaea leafsamples. The mean values of T and W are larger and that of T_osmaller than the corresponding values for free (bulk) water.The results favour a model of the organization of water in clustersrather than in multilayers and indicate a stronger binding ofwater in living systems. Key words: Dielectric relaxation, distribution of relaxation times, free and bound water     

Rehydration versus Growth-induced Water Uptake in Plant Tissues

Experiments show that the rate of water uptake by living tissues external to mature xylem of cotton stems (Gossypium hirsutum L. Auburn 7-683) is very similar to the corresponding curves for leaf tissue. In both cases one obtains a two-phase curve with phase I corresponding to passive rehydration and phase II pertaining to active growth.     

Measurement of the Water Potential of Intact Plant Tissues1

A modified design of a thermocouple psychrometer is describedwhich utilizes the Peltier effect for producing an exceedinglysmall wet-bulb thermometer. The thermocouple assembly has beenreduced to miniature dimensions and the thermocouple chamberconsists of a silver cylinder (volume 0.06 cm³) whose base isformed by the material under observation. This makes it possibleto measure water potentials over limited surfaces of intactplant tissues, e.g. germinating pea seeds (Pisum sativum L.).The apparatus is capable of measuring water potentials downto a magnitude of about –6,000 joules/kg.³     

Measurement of the Self-Diffusion Coefficient of Water as a Function of Position in Wheat Grain Using Nuclear Magnetic Resonance Imaging

A pulsed field gradient spin echo sequence has been incorporated in a nuclear magnetic resonance (NMR) imaging experiment to provide an image contrast dependent on local molecular self-diffusion. The consequent image attenuation is shown to exhibit a dependence on applied magnetic field gradient consistent with the Stejskal-Tanner relationship. The method used represents a novel extension of microscopic imaging and demonstrates the possibility of measuring localized motion.

Water self-diffusion rates normal to the transverse 1.3-mm section of a wheat grain have been measured in structural features at 150-μm resolution. The results are consistent with averaged measurements in the bulk grain obtained by other methods while local differences in water mobility correlate with differences in physiological function.

     

Anthocyanin Influence on Water Proton NMR Relaxation Times and Water Contents in Leaves of Evergreen Woody Plants During the Winter

An investigation was made to determine the effect of anthocyaninin red-colored evergreen leaves during the mid-winter on waterproton NMR relaxation times (T₁). Water contents, anthocyanincontentsand histologic localization of red-coloration in mesophyllswere determined by using both red-colored and green leaves fromthe same branches of Rhododendron, Viburnum and Mahonia, respectively.Although the decrease of water contents in the red-colored leavesin Mahonia was insignificant, decreases in the former two specieswere clearly observable. T₁ differences between red-coloredand green leaves for the three species were insignificant. Increasesof anthocyanin contents and histologic localization of red colorationin mesophylls for the red-colored leaves were more pronouncedin Rhododendron and Viburnum than in Mahonia. These observationssuggest that the pronounced increases of histologic localizationof red-colored mesophyll cells and anthocyanin contents in red-coloredleaves for the former two species contribute to maintenanceof T₁ relaxation times in spite of the marked decrease of watercontents in leaves. It is assumed that the increase of localizationareas of red-colored parenchymatous cells in mesophylls is moreeffective than the total contents of anthocyanins in leavestowards the maintenance of the T₁ level in red-colored leaves,and this appears to be dependent on the vacuolar compartmentationof anthocyanin in mesophyll cells. (Received August 3, 1991; Accepted December 12, 1991)     

Spin-lattice Relaxation in the Proton NMR of Hydrated Tobacco Cut-fillers

The spin-lattice relaxation time was measured by proton NMR of hydrated tobacco cut-fillers. The relaxation decays of adsorbed water were expressed by a single phase system below 70% relative humidity, while a two-phase system was applicable to water adsorbed at more than 80% relative humidity. From the two-phase model, it was considered that 0.12–0.13 kg water/kg dry tobacco is bound water.     

An Examination of the Refractometric Method for Determining the Water Potential of Plant Tissues

Some sources of error in refractometric determinations of waterpotential are examined and methods of correcting for them oravoiding them are discussed, as well as their relevance to Shardakov’s(Schlieren) method. The sources of the exudation from cut tissues,which causes a rise in the refractive index of water and affectsthat of solutions, are investigated. The exudation consistsmainly of sap released from cut and damaged cells. The amountof sap exuded is independent of the concentration of the externalsolution. A method of estimating the extent of the error thusincurred in water potential determinations is described. Errorarising also from the admixture of cell-wall water to the referencesolutions, lowering their effective concentration, and othersources of error are briefly described. A method of eliminating the curvature in graphs of change ofrefractive index plotted against molarity is suggested. Themethod permits the data from all concentrations to be used indetermining the null point for water exchange, instead of thosenear to the null point only.     

Estimation of the Freezing Injury in Flower Buds of Evergreen Azaleas by Water Proton Nuclear Magnetic Resonance Relaxation Times

Temperature dependence of longitudinal relaxation times (T₁)of water protons in flower buds of six azalea species differingin cold hardiness and ecological distribution was investigatedby pulse nuclear magnetic resonance spectroscopy. Thermal hysteresiswas observed for T₁ following a slow freeze-thaw cycle. TheT₁ ratio (the ratio obtained from the difference between theoriginal T₁ value in an unfrozen sample and the final T₁ aftera freeze-thaw treatment, both at 20C, divided by the originalT₁) was closely correlated with the viability of florets innon-acclimated buds of R. kiusianum. If the buds were frozento a lethal temperature and then thawed to 20C, the T₁ ratioincreased. The T₁ ratios of acclimated winter buds for the sixspecies used were correlated with the level of cold hardiness(supercooling ability of florets determined by differentialthermal analysis). The T₁ ratio of deacclimated spring buds,especially those from hardier species, markedly increased uponcooling to a lethal temperature. Species differences observedin acclimated winter buds disappeared upon deacclimation. TheT₁ ratio appears to be related to the viability of florets andthe degree of freezing damage (membrane disruption) in florets. (Received December 28, 1984; Accepted May 24, 1985)     

Measurement of the Water Potential of Intact Plant Tissues2PISUM SATIVUM L.)

Through the use of a microthermocouple psychrometer it has becomepossible to measure the water potential over part of the surfaceof a pea seed, starting about 19 hours after sowing in distilledwater, or later in the case of seeds germinating at lower osmoticpotentials, in both instances until well after radicles haveemerged. It has been shown that the potential of air-dry seedsis well below –6,000 joules/kg but increases rapidly duringimbibition, depending upon the water potential of the germinationmedium. Pea seeds subjected to lower external water potentialsgerminate at lower internal water potentials than they exhibitin distilled water. The water potentials of the seeds decreasejust after radicle emergence till the radicle establishes contactwith its germination medium, possibly as a result of demandfor moisture during that period due to incipient cell elongation.No detectable amounts of osmotically active substances are exudedfrom the seed during germination; the pea seed coat restrictsthe entry of polyethylene glycol molecules into the seed untilemergence of the radicle.     

Monitoring Stress Sensitivity by Water Proton NMR Relaxation Times in Leaves of Azaleas That Originated in Different Ecological Habitats

Responses of the leaves of five species of azalea to environmentalstresses, such as freezing, dehydration, high temperature andsalt spray, were measured in terms of water proton NMR relaxationtimes (T₁), supercooling ability, and water content. Three subtropicalspecies (R. scabrum cv. Shounoshin, R. eriocarpum and R. tashiroivar. lasiophyllum) and two northern species (R. indicum cv.Kumano-satsuki and R. yedoense f. poukhanense), which originatedin different ecological habitats, showed characteristic behaviorsin terms of T₁ relaxation times. In general, a species witha large change in T₁ is more stress-sensitive than a speciesshowing the opposite tendency. The relative sensitivity to variousstresses of each species appears to be related to the severityof conditions in its natural habitat. It seems possible thatthose species of azalea with higher sensitivity to a particularsingle stress may also exhibit higher sensitivity to severalor even most stresses, and vice versa. (Received August 27, 1992; Accepted February 26, 1993)     

Water Pathways in the Bacteriorhodopsin Proton Pump

Internal water molecules play key roles in the functioning of the light-driven bacteriorhodopsin proton pump. Of particular importance is whether during the proton-pumping cycle the critical water molecule w402 can relocate from the extracellular to the cytoplasmic side of the retinal Schiff base. Here, classical mechanical and combined quantum mechanical/molecular mechanical reaction path computations are performed to investigate pathways and energetic factors influencing w402 relocation. Hydrogen bonding between w402 and the negatively charged Asp85 and Asp212 largely opposes repositioning of the water molecule. In contrast, favorable contributions from hydrogen bonding of w402 with the Schiff base and Thr89 and from the untwisting of the retinal polyene chain lower the energetic cost for water relocation. The delicate balance between the competing contributions underlies the need for highly accurate calculations and structural information.     

A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues

A microscale technique has been developed for routine quantifications of picogram amounts of indole-3-acetic acid (IAA) in plant tissues by combined gas chromatography-mass spectrometry. Low- and high-resolution selected-ion-monitoring and selected-reaction-monitoring mass spectrometry techniques were compared for selectivity and precision. The best selectivity was obtained with selected-reaction-monitoring analysis, and 1-mg samples containing 500 fg of IAA could be analyzed accurately with this method. This technique was used to investigate the IAA distribution pattern along the longitudinal axis of tobacco (Nicotiana tabacum [L.]) leaves. In young, developing leaves an increase of endogenous IAA from the leaf tip to the base of the leaf was observed, whereas the level of IAA was uniform along this axis in mature leaves.     

The Physiological Significance of Mitochondrial Proton Leak in Animal Cells and Tissues

Mitochondrial proton leak is an important component of cellular metabolism in animals and may account for as much as one quarter to one third of the Standard Metabolic Rate of the rat. The activity of the proton leak pathway is different in a wide range of animal species and in different thyroid states. Such differences imply some function for proton leak and candidates for this function include thermogenesis, protection against reactive oxygen species, endowment of metabolic sensitivity and maintenance of carbon fluxes.     

Regulators of Cell Division in Plant Tissues

Procedures were devised for purification of the cytokinins in the milk of mature coconuts (Cocos nucifera fruits). One cytokinin isolated in crystalline form was unequivocally identified as 9-β-D-ribofuranosylzeatin. This compound appeared to account for a large proportion of the cytokinin activity in n-butanol extracts of coconut milk. The activity which was not extracted by n-butanol was largely due to unidentified compounds which could be partially purified by adsorption onto and elution from charcoal.