Translational and rotational motions of proteins in a protein crowded environment

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.     

Transient electric birefringence of colloidal particles immersed in shear flow. Part II. The initial response under the action of a rectangular electric pulse and the behavior at a low alternating electric field

The time-dependent rotational diffusion equation for rigid macromolecules in solution has been approximately solved for two cases in order to extend the electric birefringence technique to streaming-electric birefringence. One is for the initial period through the application of a rectangular electric pulse to the solution immersed in a low shear flow. The purpose of this is expansion of the distribution function into a function series made by the product of the powers of reduced time (= Thetat) and hydrodynamic field alpha (= G Theta , G: velocity gradient, Theta: rotary diffusion constant) and a surface harmonic P(i)(j)cos jphi. The solution for the build-up process at arbitrary electric field strength is found, but is limited to low hydrodynamic fields. The other is for the response when an alternating electric field is applied to the solution in a shear flow. Here, instead of reduced time, the maximum electric field E(0) is chosen as a parameter for the expansion. The expressions for the intensity of the transmitted light through crossed Nicols are derived in two optical systems where the polarizer is set at an angle of 45 degrees and 0 degrees to the direction of the electric field. The results in the former case show that we can determine four parameters, the ratio of velocity gradient to rotary diffusion constant, the axial ratio of a particle, the anisotropy of electric polarizability, and the optical anisotropy factor, from four values observed in two optical systems, namely, two light intensities before applying an electric field and two initial slopes of the build-up after applying an electric field. On the other hand, when a low alternating electric field with extremely high frequency is applied, the build-up of the light intensity in the former case is the same as that of electric birefringence for pure induced dipole orientation. The build-up for the latter optical system is the same as the expression for pure induced dipole orientation of Eq. (38) shown in a previous work.     

Transient electrical birefringence characterization of heavy meromyosin

Heavy meromyosin (HMM) and myosin subfragment 1 (S1) were prepared from myosin by using low concentrations of alpha-chymotrypsin. The light chain distribution in HMM was identical with that of myosin, within experimental error, when analyzed on 12% polyacrylamide gels after electrophoresis. Specific birefringences and birefringence decay times were measured by transient electrical birefringence in 5 mM KCl, 5 mM tris(hydroxymethyl)aminomethane (pH 7), and 1 mM MgCl2 at 4 degrees C under gentle conditions that reduced the CaATPase activity by less than 10%. For solutions of HMM, by use of electric field pulses shorter than 0.5 microseconds, the birefringence decay signal from the S1 portions of HMM could be resolved and the rotational motions of the S1 moieties observed directly. The rotation relaxation time, adjusted to 20 degrees C, was 0.34 microseconds; this is in quantitative agreement with previous hydrodynamic results obtained by using covalently attached probes. The assignment of the fast decay time obtained with HMM to the S1 portions was confirmed by birefringence decay measurements on free S1, for which the relaxation time was 0.13 microseconds, corrected to 20 degrees C. The specific birefringences for S1 and HMM, respectively, were 0.37 X 10(-6) and 12.8 X 10(-6) (cm/statvolt)2. Thus, for much longer electric field pulses, the signal from HMM is due almost entirely to its subfragment 2 (S2) portion, and its rotational dynamics can also be monitored directly by using electrical birefringence. The decay of the signal from the S2 portion could be adequately fit without evoking bending of the S2 portion of HMM other than at its junction with S1.     

Ligand-induced myosin subfragment 1 global conformational change

The effects of selected ligands on the structure of myosin subfragment 1 (S1) were compared by using transient electrical birefringence techniques. With pairs of dilute solutions of S1 at 3.5 degrees C in low ionic strength (mu = 0.020 M) buffers that had matched electrical impedances, S1 with Mg2+, MgADP, or MgADP.Vi bound was subjected to 6-7-microseconds external electrical fields in the Kerr law range. Specific Kerr constants and the rates of rotational Brownian motion after the electric field was removed were measured. Neither Mg2+ nor MgADP had a measurable effect on either observable, but when orthovanadate (Vi) bound S1.MgADP it decreased the rotational correlation coefficient from 267 +/- 6 to 244 +/- 10 ns. Parallel measurements of MgATPase activity indicated that S1.MgADP.Vi was greater than 95% inhibited. These results confirm the conclusion of Aguirre et al. [(1989) Biochemistry 28, 799] that Vi binding to S1.MgADP increases its rate of rotational Brownian motion and provide data that are more quantitatively correlated with S1 structure. The Vi-induced change in the rotational correlation coefficient is consistent with S1 becoming more flexible or more compact when Vi binds. Assuming that S1.MgADP.Vi is an analogue for S1.MgADP.Pi, the structural changes observed for S1-ligand complexes in solution are discussed in relation to possible structural changes of intermediates on the kinetic pathway of ATPase hydrolysis. A new model of force generation by S1 in muscle is hypothesized.     

A hydrodynamic study of collagen fibrillogenesis by electric birefringence and quasielastic light scattering

Neutral soluble collagen was extracted from lathyritic rat skin under proteolysis-inhibited conditions. Purified solutions were characterized by electric birefringence and heterodyne beat quasi-elastic light-scattering techniques under conditions where the monomeric form was stable (at 4 degrees C in 0.032 M phosphate buffer at pH 7.04). Solutions were then heated and the birefringence and light scattering followed during the fibrillogenesis reaction. The monomer presents a translational diffusion coefficient of 0.85 X 10(-7) cm2/s and a rotary diffusion coefficient of 1150 +/- 50 s-1; these values are consistent with a rodlike molecular model of 220 +/- 10 nm length and 4 +/- 1 nm diameter, substantially different from electron microscopic values of 290 and 1.5 nm, respectively. We propose that at pH 7.04 and relatively high ionic strength, the collagen monomer unit must exhibit substantial deviation from a completely rigid and extended rodlike structure. During the entire lag phase in a thermally induced fibrillogenesis reaction, the relaxation times for both translational and rotational motion remain virtually unchanged. The monomer polarity is also unchanged, as shown by reverse pulse birefringence data. No intermediate size soluble aggregates, such as dimers or trimers, have been detected between monomer and very large aggregates or fibrils during the process, although early multistep assembly products (dimers, trimers) could have been seen if present. These data suggest a model for fibrillogenesis emphasizing a monomer-related nucleation event, such as internal stiffening or conformational transition, followed by a rapid continuous growth up to large fibrils.     

Transient electric birefringence study of the length and stiffness of short DNA restriction fragments

Transient electric birefringence measurements of the rotational diffusion constant of five short restriction fragments of the plasmid pBR322 show that the hydrodynamic length is independent of sodium ion concentration in the range of 0.2 to 2.5 mM. The fragments are too stiff to be modeled as wormlike molecules. The rotational relaxation times of the fragments, which range from 64 to 124 base pairs, have been used to calculate the rise per base pair using six different theoretical expressions for the length dependence of the rotational diffusion coefficient of straight cylinder. The best estimate for the rise per base pair of Na-DNA in solution is 3.3 ± 0.1 Å.     

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.     

Rotational diffusion of short DNA fragments in polyacrylamide gels: an electric birefringence study

Using electric birefringence we have examined the rotational diffusion of five short DNA fragments (55 to 256 base pairs) both in polyacrylamide gels as a function of gel concentration and in solution. The length dependence of the measured rotational relaxation times in the gels is in good agreement with the prediction from the Odijk theory for the dynamics of slightly flexible rods in a network. The rotational relaxation times were found to depend on the gel concentration, contrary to the prediction from the Odijk theory. Possible reasons for this observation are discussed. The birefringence decay curves for DNA fragments in the gel were single exponential only at small electric field strength.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

Diffusion of lysozyme chloride in water and aqueous potassium chloride solutions.

The diffusion of lysozyme chloride in aqueous solution has been studied at 25 degrees C using the Goüy interferometric technique. The concentration dependence of the diffusion coefficient in water has been measured over the concentration range 1.1599-9.1556 gcm-3 and the results suggest a value of D 25, w at infinite dilution of 5.838 x 10(-6) cm2s-1. The variation in diffusion coefficient with ionic strength has also been considered by following the diffusion of 0.45% lysozyme chloride in a series of potassium chloride solutions. The value of D in 0.15 M KCl has been found to be approximately one quarter of that in water alone an the diffusion coefficient has been shown to increase markedly as the KCl concentration is reduced below 0.05 M. Interpretation of these observations involves consideration of solution electrostatic effects.     

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.     

Tracer diffusion coefficients of proteins by means of holographic relaxation spectroscopy: application to bovine serum albumin

Holographic relaxation spectroscopy has been used to measure tracer diffusion coefficients for photochromically labeled bovine serum albumin in solutions having total bovine serum albumin concentrations in the range 3.25 to 257 g/liter. In the limit of zero concentration, the diffusion coefficient (20 degrees C, 0.1 M NaCl, 0.05 M Tris, pH 8.0) was found to be (5.9 +/- 0.1) X 10(-7) cm2/s and the initial slope was zero. The concentration dependence of the diffusion coefficient was not significantly affected by the fraction of protein molecules which were labeled. Holographic relaxation spectroscopy permits rapid, accurate determination of tracer diffusion coefficients for proteins in mixtures.     

Nucleotide- and temperature-induced changes in myosin subfragment-1 structure.

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.     

Transient electric birefringence study of highly dilute agarose solutions

In order to characterize the first step of agarose gelation, highly dilute solutions (2·10^?3 to 0.5 g/L) have been studied by means of the transient electric birefringence technique. The field-free decay curves of the birefringence are well described by a stretched-exponential B(t) ≈ exp(?t/τ)^β; the value of the exponent β is close to 0.5 whatever the agarose concentration. The suspended particles observed by electron microscopy present a rod-like shape with a constant diameter (～50 Å), without any branching; they are polydisperse with a distribution of lengths approximately exponential. The mean length of these fibers, deduced from their mean rotational diffusion coefficient, is proportional to the 0.37 power of the agarose concentration in the solution. Furthermore, these particles possess a strong permanent electrical dipole confirming the side-to-side arrangement of helices into bundles; this dipole is roughly proportional to the particle length, indicating a self-similarity in the unidirectional growth of the agarose fibers, even when approaching the gelling concentration.     

DNA persistence length revisited

DNA restriction fragments ranging from 79 to 789 base pairs in length have been characterized by transient electric birefringence (TEB) measurements at various temperatures between 4 and 43 degrees C. The DNA fragments do not contain runs of four or more adenine residues in a row and migrate with normal electrophoretic mobilities in polyacrylamide gels, indicating that they are not intrinsically curved or bent. The low ionic strength buffers used for the measurements contained 1 mM Tris Cl, pH 8.0, EDTA, and variable concentrations of Na(+) or Mg(2+) ions. The rotational relaxation times were obtained by fitting the TEB field-free decay signals with a nonlinear least-squared fitting program; the decay of the birefringence was monoexponential for fragments < or = 241 base pair (bp) in length and multiexponential for larger fragments. The terminal relaxation times, characteristic of the end-over-end rotation of the DNA molecules, were then used to determine the persistence length (p) and hydrodynamic radius (r) of DNA as a function of temperature and ionic strength, using several different hydrodynamic models. The specific values obtained for p and r are model dependent. The wormlike chain model of P. J. Hagerman and B. H. Zimm (Biopolymers 1981, Vol. 20, pp. 1481-1502) combined with the revised Broersma equation (J. Newman et al., Journal of Mol Biol 1997, Vol. 116, pp. 593-606) appears to be the most suitable for describing the flexibility of DNA in low ionic strength solutions. The values of p and r obtained from the global least squares fitting of this equation are independent of DNA length, and the deviations of the individual values from the average are reasonably small. The consensus r value calculated for DNA in various low ionic strength solutions containing 1 mM Tris buffer is 14.7 +/- 0.4 A at 20 degrees C. The consensus p values decrease from 814 approximately 564 A in solutions containing 1 mM Tris buffer plus 0.2-1 mM NaCl and decrease still further to 440 A in solutions containing 0.2 mM Mg(2+) ions. The persistence length exhibits a shallow maximum at 20 degrees C and decreases slowly upon either increasing or decreasing the temperature, regardless of the model used to fit the data. By contrast, the consensus values of the hydrodynamic radius are independent of temperature. The calculated persistence lengths and hydrodynamic radii are compared with other data in the literature.     

Myosin subfragment 1 has tertiary structural domains

Transient electrical birefringence measurements were made on skeletal muscle myosin subfragment 1 (S1) at 3.7 degrees C in 10 mM tris(hydroxymethyl)aminomethane-acetate and 0.10 mM MgCl2, pH 7.0. The specific birefringence for 4.5 microM S1 was determined from steady-state measurements to be (8.1 +/- 0.3) X 10(-7) (cm/statvolt)2. For electric fields in the range of 2.47-24.7 statvolts/cm, the alignment was due to a large permanent dipole moment for S1, estimated to be 8500 +/- 2000 D. The duration and the strength of the transient electric field was varied, and the temporal response of the decay of the birefringence signal was analyzed. The rate of rotational motion after the field was removed increased with increasing field strength for short (0.35-microseconds) pulses and decreased with increasing pulse lengths for all field strengths. The rate of decay from a steady-state birefringence signal was independent of field strength. A model of S1 structure is proposed, which is consistent with these data and most other data on S1 structure. In this model, S1 is composed of two tertiary structural domains that are connected by a flexible linkage with a substantial restoring force. The electric dipole moments on the two domains are arranged head to tail. The segmental movement of the domains is restricted to certain directions. The average conformation of the molecule is elongated, but it can be made more compact by the torque exerted by an electric field. The structural changes depend on the strength and duration of the pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

The flexibility of A-form DNA.

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Dielectric behavior of polyelectrolytes. VI. Dynamic response of cylindrical biopolymers to electric fields

The time dependence of the orientation of a cylindrical biopolymer and the configuration of its counterion complement in the presence of an external electric field is found by solving a model forced diffusion equation. The solution is a high temperature expansion in the external field strength and is used to predict the nature of the dielectric relaxation and the dynamic Kerr effect for such systems. Specific application is made to the dynamic Kerr effect of a DNA oligomer for which experimental data appear in the literature. The analysis yields a value for the surface diffusion coefficient of a sodium ion on DNA at 20 degrees C of 3.8 x 10(-10) m2 s-1.