NMR relaxation processes of 31P in macromolecules

³¹P-Nmr relaxation parameters (spin-lattice relaxation time, linewidth, and nuclear Overhauser effect) were obtained at three different frequencies for poly(U) and a well-defined (145 ± 3 base-pair) fragment of DNA in solution. Data sets for the two samples were analyzed by theories which included relaxation by the mechanisms of ³¹P chemical shift anisotropy as well as by ¹H-³¹P dipole–dipole interaction. Neither data set could be satisfactorily described by a single correlation time. A model of a rigid rotor most nearly fits the data for the DNA molecule. Parameters obtained from the least-square fit indicate (1) that the DNA undergoes anisotropic reorientation with a correlation time τ₀ = 6.5 × 10^?7 sec for the end-to-end motion, (2) the ratio of diffusion constants D_∥/D_⊥ is 91, and (3) that the linewidth is due to chemical shift dispersion to the extent of 0.5 ppm. Some deviations of the calculated from the observed values suggested that significant torsional and bending motions may also take place for this DNA. Another model which contains isotropic motion but with a broad distribution of correlation times was required to fit the data for poly(U). A log ? χ² distribution function of correlation times [Scheafer, J. (1973) Macromolecules 6 , 881–888] described well the motion of poly(U) with the average correlation time τ = 3.3 × 10^?9 sec and a distribution parameter p = 14.     

Rotational relaxation of macromolecules determined by dynamic light scattering. I. Tobacco mosaic virus

The intensity autocorrelation function for the depolarized component of forward-scattered light from a solution of large polymeric molecules is derived in terms of the correlation function for the amplitudes of the Y₂,±₁(θ,?) fluctuations in the anglar distribution of segments in the solution without any assumptions regarding the statistical properties of the scatterad light field. Effects arising from the use of polychromatic incident light and from the mixing of the scattered and polychromatic incident light beams are examined in detail. Apparatus for observing the depolarized forward-scattered light, digitizing and storing the fluctuating phototube current at rates from 10 to 540,000 times per second, and computing the correlation functions directly in the time-domain is described herein. Correlation functions were obtained for 0.05 mg/ml solution of tobacco mosaic virus at pH 9.1 and also at pH 6. The degree of association of the virus appears to be independent of pH, and the monomer relaxation times (corrected to 25°C) extracted from the data by a least-squares procedure lie in the range 0.44–0.49 msec, also independent of pH. The absence of faster component in the correlation function between 6 μsec and 0.5 msec is used in conjunction with thermal fluctuation theory to infer a lower limit for the effective Young's modulaus of the rod, E ≤ 2.5 × 10⁷ dynes/cm².     

Rotational relaxation of macromolecules determined by dynamic light scattering. II. Temperature dependence for DNA

Correlation functions have been determined for the fluctuating intensity of the depolarized component of forward-scattered laser light from solutions of DNA. The molecular correlation function of calf thymus DNA (mol wt ～15 × 10⁶) appears to exhibit a longest relaxation time (τ_25,w, ～ 18 msec) close to what one would predict from the flowdichroism measurements of Callis and Davidson and, in addition, manifests a spectrum of faster times down to tenths of milliseconds. Furthermore, a major fraction of the amplitude of fluctuations in the angular distribution of segment axes is relaxed on a very much shorter time scale (of the order of 20 microseconds) that appears to be relatively insensitive to molecular weight of the DNA, or to near-melting temperatures. The temperature profile of the longest relaxation time has been obtained and found to exhibit a peculiar spike near T_m, which, together with the absence of a corresponding spike in the (high shear) viscosity, has been interpreted as indicative of an increase in the molecular weight of the DNA in a narrow temperature region near T_m. Correlation functions for polarized light scattered at finite angles were obtained in an attempt to determine the temperature dependence of the translational diffusion coefficient. Although the data contain an extremely slow component that does not admit a simple interpretation, there is some indication of a decrease in the translational diffusion coefficient near T_m, thus supporting the notion of an aggregation occurring near T_m. Finally, a “counterion escape” mechanisn is proposed for the apparent aggregation.     

E-type delayed fluorescence depolarization, technique to probe rotational motion in the microsecond range.

E (eosin)-type delayed fluorescence depolarization studies extend the time range for the measurement of rotational diffusion to microseconds and ms, thereby allowing investigation of slow rotational movement of macromolecules like membrane proteins. An apparatus is described for the determination of time-dependent anisotropy in this interesting time range. The method has been tested on eosin-labelled cytochrome P-450 incorporated into phospholipid membrane vesicles.     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

The dynamic range problem in the analysis of the plasma proteome

One of the greatest challenges in analyzing the plasma proteome is the wide range of concentration of different proteins. The current study examines the range of protein concentration for 18 proteins measured over a year in a clinical laboratory to provide data on pathological extremes in protein concentrations. The complete measured range, from upper limits for albumin to lowest values for thyroid-stimulating hormone (TSH), represented more than 10 logs of molar abundance. A number of plasma proteins measured in the clinical laboratory varied over a concentration range spanning more than 4 logs, and limits of detection of clinical assays were inadequate to assess full concentration ranges of several proteins. Considering reported values from studies using higher sensitivity assays suggest that plasma concentrations of prostate-specific antigen (PSA), human chorionic gonadotropin (hCG), and cardiac troponin I vary by more than 7 logs. All of the plasma proteins measured in the present study represent secretory proteins or highly expressed components of specific tissues. Thus, the dynamic range for these components is likely to greatly underestimate the total range of protein concentration in the plasma proteome.     

Digital dynamic range expansion applied to X-ray densitometric analysis of total hip replacement

Due to the presence of the stiff prosthetic stem fitted in the medullary canal during total his replacement, the surrounding cortex of the femur changes its density over time. This bone remodelling takes place with every type of total hip prosthesis; however, its intensity may vary between prostheses and patients. In the worst cases this process can lead to the late failure of the implant. To monitor such bone density evolution, we are developing a tailored Computer-aided Densitometric Image Analysis system (the major part of this our system uses an 8-bit commercial hardware with 256 levels of grey). The equivalent dynamic range of an X-ray picture is about 10 bits. In this paper we present a method to overcome these hardware limitations by improving the software. Using a double-exposure acquisition it is possible to build a 9-bit image that is good enough for most applications involving bone density measurement.     

31P Magnetic relaxation studies of yeast transfer RNAPhe.

The molecular mechanism of thermal unfolding of yeast tRNA^Phe in 20 mM NaCl, 1 mM EDTA, and 10 mM MgSO₄, pH 7.1 ± 0.1, has been examined by ³¹P magnetic relaxation and the nuclear Overhauser effect methods at 40.48 MHz in the temperature range of 22.5–80°C. Two partially resolved ³¹P resonance peaks of yeast tRNA^Phe have been found to behave distinctively different in their longitudinal relaxation times. Individual intensities of the two partially resolved peaks have been quantitatively estimated by the use of relaxation data and the nuclear Overhauser effect as a function of temperature. The results of these observations largely support the earlier suggestion by Guéron and Shulman that the high- and low-field parts of the main ³¹P resonance cluster originate from phosphorus nuclei belonging to the double-helical and nonhelical regions of the tRNA, respectively. The spin-lattice relaxation of the phosphorus nucleus has been found to be determined dominantly by the dipolar interaction with the surrounding ribose protons at this observing frequency. Rotational correlation times for the two portions of the ribose-phosphate backbone of the tRNA have been separately deduced from the quantitative treatment of the ³¹P nuclear spin-lattice relaxation times (T₁) and the nuclear Overhauser effect. The result indicates that the two portions undergo internal motions at distinctively different rates of 10⁸–10¹⁰ sec^?1 order in the temperature range of 22.5–80°C, and that the thermal activation of these motions occurs at least in three distinctive steps, i.e., 22.5–31, 31–40, and 40–80°C. The rates of the internal motions and the associated activation energies in respective steps give some insight into the thermo-induced change of the yeast tRNA^Phe structure.     

Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes.

The theory of fluorescent emission anisotropy [r(t)] of a cylindrical probe in a membrane suspension is developed. It is shown, independent of any model, that the limiting anisotropy [r(infinity)] is proportional to the square to the order parameter of the probe. The order parameter determines the first nontrivial term in the expansion of the equilibrium orientational distribution function of the probe in a series of Legendre polynomials. Following Kinosita, Kawato, and Ikegami, the motion of the probe is described as diffusion ("wobbling") within a cone of semiangle theta 0. Within the framework of this model, an accurate single-exponential approximation for r(t) is considered. An analytic expression relating the effective relaxation time, which appears in the above approximation, to theta 0 and the diffusion coefficient for wobbling is derived. The model is generalized to the situation where the probe is attached to a macromolecule whose motion cannot be neglected on the time scale of the fluorescence experiment. Finally, by exploiting the formal similarity between the theory of fluorescence depolarization and 13C-NMR dipolar relaxation, expressions for T1, T2, and the nuclear Overhauser enhancement are derived for a protonated carbon which is nonrigidly attached to a macromolecule and undergoes librational motion described as diffusion on a spherical "cap" of semiangle theta 0.