Acid-induced structural changes of a mouse IgG2a monoclonal antibody (MN12) studied by transient electric birefringence measurement.

Acid-induced structural changes of a mouse IgG2a monoclonal antibody (MN12) as indicated by Jiskoot et al. (Eur. J. Biochem. 201,223-232 (1991)) were studied by measuring the transient electric birefringence of MN12 in aqueous solution and in glycerol-water mixtures at different pH conditions. A multi-exponential analysis program, DISCRETE (Provencher,S.W., Biophys.J.16,27-41 (1976)), and a constrained inverse Laplace transform program, CONTIN (Provencher, S.W., Comp. Phys. Comm. 27, 213-227 (1982)) have been used to determine the number of exponentials needed to represent the data and their decay times. Measurement of the time-resolved electric field induced birefringence makes it possible to study rotational processes on a timescale from several tens of nanoseconds to microseconds. This enabled us to monitor the segmental flexibility and the rotational motion of single antibody molecules as well as the occurrence of aggregates. The results show an increase in hydrodynamic dimensions of MN12 upon lowering the pH from 6.6 to 2.7. Additionally, the original segmental flexibility, which could be monitored for the samples in glycerol-water mixtures, is altered at low pH. The results have been interpreted as swelling of MN12 followed by dimerization.     

Non-random conformation of a mouse IgG2a monoclonal antibody at low pH

The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 microseconds) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 microseconds; a further increase to 0.30 microseconds was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.     

Solvent-exposed tryptophans probe the dynamics at protein surfaces.

The dynamics of single tryptophan (W) side chain of protease subtilisin Carlsberg (SC) and myelin basic protein (MBP) were used for probing the surface of these proteins. The W side chains are exposed to the solvent, as shown by the extent of quenching of their fluorescence by KI. Time-resolved fluorescence anisotropy measurements showed that the rotational motion of W is completely unhindered in the case of SC and partially hindered in the case of MBP. The rotational correlation time (phi) associated with the fast local motion of W did not scale linearly with the bulk solvent viscosity (eta) in glycerol-water mixtures. In contrast, phi values of either W side chains in the denatured proteins or the free W scaled almost linearly with eta, as expected by the Stokes-Einstein relationship. These results were interpreted as indicating specific partitioning of water at the surface of the proteins in glycerol-water mixtures.     

Electric and hydrodynamic properties of polypeptides in solution. II. Conformation of poly(L-glutamic acid) in various organic solvents

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol, dimethyl sulfoxide, dimethylformamide, N-methylacetamide, trifluoroacetic acid, dioxane–water mixtures (3:1 and 4:1 by volume), and dioxane–formamide mixture (1:1 by volume) has been measured by the use of the rectangular pulse technique at 30 °C. The intrinsic viscosity has also been measured at the same temperature. The magnitude of the specific Kerr constant and the intrinsic viscosity suggests that PLGA is helical and has a large dipole moment in methanol, dimethyl sulfoxide, dimelhylformamide, N-methylacetamide, and dioxane–water mixtures. In this case we have obtained the length distribution curve and the mean length of PLGA molecules from the decay of the electric birefringence, by applying the method recently developed for helical polypeptides. Furthermore, we have proposed and applied a method of obtaining the mean dipole moment and the optical anisotropy factor from the field strength dependence of the electric birefringence for polydisperse systems on the basis of the knowledge on the length distribution. The results show that PLGA may have a different helical conformation in dimethyl sulfoxide. The specific Kerr constant of PLGA in trifluoroacetic acid is very small, which suggests that PLGA is a random coil in this solvent.     

Rigidity of myosin and myosin rod by electric birefringence

The rotational relaxation times of rabbit myosin and myosin rod have been determined by electric birefringence measurement. The relaxation time of myosin measured in 10 mM pyrophosphate buffers in a pH range of 7.6–9.5 was found to have substantial concentration and pH dependences. The infinite-dilution limit of the relaxation time, τ°, was determined as 38 ± 2 μs, and it was found to be independent of pH. For myosin rod, a possible thermally induced conformational change was investigated in a temperature range of 1–43°C. The rotational relaxation time of myosin rod shows no clear indication of conformational change in this temperature range, and the radius of gyration measurement by light scattering was shown to be consistent with this observation. The steady-state birefringence, however, decreases substantially above around 40°C. This, the myosin rod appears to be only slightly flexible even at physiological temperature, but the possibility of a “melting” or “hinging” of the myosin rod cannot completely be ruled out on the basis of these experiments.     

Orientational interactions in low-concentration DNA solutions

The rotational relaxation tiem τ₃ of DNA molecules (M_w ? 5 × 10⁶) in solution has been determined by the transient electric birefringence method. The analysis of the birefringence decay makes it possible to study only the higher-molecular-weight fraction, the molecules being considered as rigid elongated particles in a short time scale. A marked concentration dependence of the relaxation time has been observed for DNA in low ionic strengths. Above a critical concentration c*, τ₃ increases with the DNA concentration, c. The value of c* increases with the ionic strength. For 10^?3 ionic strength (with NaCl), c* is about 10 μg/mL; then we observe the same strong concentration dependence of rotational relaxation times as recently reported for rodlike M-13 viruses [Maguire, J. F., McTague, J. P. & Rondelez, F. (1980) Phys. Rev. Lett. 45 , 1891–1894]. These results may be discussed in terms of the Doi-Edwards theory for rotational relaxation time of rigid macromolecules [Doi, M. (1975) J. Phys. 36 , 607–611; Doi, M. & Edwards, S. F. (1978) J. Chem. Soc. Faraday Trans. 74 , 918–932] and the critical concentration above which the interactions between the molecules begin to appear allows determining the corresponding molecular length. We observe a very good agreement between the DNA lengths obtained from the c* values and by using the infinite dilution value of τ₃ and Broersma's equation. Therefore, only highly diluted solutions can be used if intrinsic molecular properties based on the rotational diffusion of high-molecular-weight elongated molecules are studied.     

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.     

Birefringence of Protein Solutions and Biological Systems: II. Studies on TMV, Tropocollagen, and Paramyosin

The intrinsic birefringences of TMV, tropocollagen, and paramyosin were calculated from flow birefringence measurements using the theory of Peterlin and Stuart. The values are -0.029, -0.029, and -0.030, respectively. The intrinsic birefringences of TMV and tropocollagen were measured as a function of the refractive index of the solvent in glycerol-water mixtures. In both cases the values were not constant and became less negative as the refractive index increased. Theoretical calculations showed that the large solvent effect could not be caused by a hydration shell of index different from that of the bulk solvent. It is concluded that either (a) the intrinsic birefringence calculated from the Peterlin-Stuart theory is incorrect or (b) the intrinsic birefringence depends markedly on the solvent. These results are of importance to the problem of quantitative polarized light microscopy since the separation of form and intrinsic birefringence contributions is based on the assumption that intrinsic birefringence is independent of solvent.     

Light chain-dependent myosin structural dynamics in solution investigated by transient electrical birefringence.

The technique of transient electrical birefringence was used to compare some of the electric and structural dynamic properties of myosin subfragment 1 (S1(elc, rlc)), which has both the essential and regulatory light chains bound, to S1(elc), which has only an essential light chain. The rates of rotational Brownian motion indicate that S1(elc, rlc) is larger, as expected. The permanent electric dipole moment of S1(elc, rlc) is also larger, indicating that the regulatory light chain portion of S1(elc, rlc) has a dipole moment and that it is aligned head-to-tail with the dipole moment of the S1(elc) portion. The permanent electric dipoles decrease with increasing ionic strength, apparently because of ion binding to surface charges. Both S1(elc, rlc) and S1(elc) have intrinsic segmental flexibility, as detected by the ability to selectively align segments with a brief weak electric field. However, unlike S1(elc), which can be structurally distorted by the action of a brief strong electric field, S1(elc, rlc) is stiffer and cannot be distorted by fields as high as 7800 V/cm applied to its approximately 8000 D permanent electric dipole moment. The S1 . MgADP . Pi analog S1 . MgADP . Vi is smaller than S1 . MgADP, for both S1(elc, rlc) and S1(elc). Interestingly, the smaller, stiffer S1(elc, rlc) . MgADP . Vi complex retains intrinsic segmental flexibility. These results are discussed within a framework of current hypotheses of force-producing mechanisms that involve S1 segmental motion and/or the loss of cross-bridge flexibility during force production.     

Electric and hydrodynamic properties of polypeptides in solution. IV. A new conformational change of poly(L-glutamic acid) in dimethylsulfoxide–methanol mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in dimethylsulfoxide (DMSO)–methanol mixtures has been measured by use of the rectangular pulse technique. The length distribution curve, the mean molecular length, and the mean apparent permanent dipole moment of PLGA in solution have been obtained from the decaycurve and field strength dependence of the steady-state birefringence according to the method developed for analyzing the electric birefringence of a polydisperse system. The length distribution curve exhibits one or two peaks. The length corresponding to a high peak and the mean length of PLGA undergo an abrupt change in the vicinity of 50 to 60 vol % DMSO at 30°C. Moreover, a sharp change of the Moffitt b₀ parameter with the solvent composition is observed. These results provide evidence for the existence of a solvent-induced transition from a helical conformation (presumably α-helix) to another helical conformation with shorter length per amino acid residue. Further, the temperature dependence of the length distribution of PLGA in 50 vol % DMSO suggests the existence of a temperature-induced helix ? helix transition.     

The Flexibility of A-Form DNA

Abstract

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.     

Liposomal heme as oxygen carrier under semi-physiological conditions orientation study of heme embedded in a phospholipid bilayer by an electrooptical method

The meso-tetra(α,α,α,α(o-pivalamidophenyl))porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37°C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.     

Electrical and hydrodynamical properties of polypeptides in solution. I. Poly(L-glutamic acid) in methanol-water mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol–water mixtures has been measured by the use of the rectangular pulse technique at 25°C. The permanent dipole moment, the anisotropy of electrical polarizability, and the optical anisotropy factor of PLGA in solution were obtained from the dependence of the steady-state birefringence on the electric field strength. Further, the mean length of PLGA in solution was calculated by a parameter method developed for analyzing the decay curve of electric birefringence. The permanent dipole moment per unit length obtained from these studies was 2.9₆, 2.4₈, 2.3₀, 2.6₆ D/Å in pure methanol, 10, 30, and 50 vol-% water, respectively. The increase of water content caused the decrease of the mean length and broadened the length distribution of PLGA. These results are discussed in relation to the viscosity and the electrical conductivity of PLGA solutions.     

Electric Birefringence of Dilute Agarose Solutions

Abstract

The technique of transient electric birefringence was used to investigate the orientation of agarose solutions in pulsed electric fields. If the agarose was dissolved in deionized water, the sign of the birefringence was positive when the electric field was small, indicating that the agarose molecules were orienting parallel to the electric field lines. The decay of the birefringence was rapid, consistent with the orientation of individual agarose helices. The amplitude of the birefringence, but not the birefringence decay times, increased as the agarose solution aged, suggesting that the helices formed slowly from the sol state. Increasing the amplitude or duration of the pulsed electric field caused additional negative, and then positive, birefringence signals to appear, characterized by much slower rise and decay times, consistent with the formation of aggregates. The slowest decay times ranged from 7.5–9.0 s, suggesting that the aggregates were several microns in size. When agarose was dissolved in dilute Tris buffer instead of deionized water, the fast positive birefringence signal was not observed, suggesting that individual helices were not present in solutions containing dilute buffer.     

Monte Carlo approach to the analysis of the rotational diffusion of wormlike chains

A Monte Carlo analysis is presented which establishes a relationship between the rotational diffusion coefficients and the flexibility (persistence length, P) of short, wormlike chains. The results of this analysis are presented in terms of experimentally observable quantities; namely, the rotational relaxation times for the field-free decay of optical anisotropy. The pertinent theoretical quantity is R, defined as the ratio of the longest rotational relaxation time of a wormlike chain to the transverse rotational relaxation time of a rigid cylinder having the same axial length (L) and segmental volume. R, so defined, is essentially independent of the axial ratio of the cylinder for any value of L/P within the range of validity of the present analysis (axial ratio > 20; 0.1 < L/P < 5). It is pointed out that P can be determined with reasonable accuracy even in the absence of a precise knowledge of the local hydrodynamic radius of the chain.     

Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

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)     

Spin-label studies of the pH-induced random coil to α-helix conformational transition in poly(L-lysine)

Poly(L -lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L -lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L -lysine) is deprotonated. For the high-molecular-weight poly(L -lysine) this corresponds to the formation of the α-helix and indicates that the side chain–side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L -lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain–side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L -lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.     

Secondary structure of globulins from plant seeds: a re-evaluation from circular dichroism measurements

The secondary structure parameters of plant seed globulins (11S from Brassica napus L, 11S from Helianthus annuus L, IIS from Vicia faba, 7S from Phaseolus vulgaris L) have been determined from their circular dichroism spectra by the method of Provencher and Glöckner. According to this method, the proteins contain 40–50% β-sheet structure and only about 10% helical structure. We conclude, therefore, that the plant seed globulins belong to the class of β-sheet proteins. Their overall secondary structure is homologous. It is shown that the method of Provencher and Glöckner provides reasonable secondary structure parameters for proteins which are rich in β-sheet structure even if the spectral range utilized for analysis is restricted to 210–240 nm.