Dynamic structure of human serum transferrin from transient electric birefringence experiments.

In order to investigate the secondary, tertiary, and dynamic structure of the iron-free (apo) and iron-saturated (holo) forms of human serum transferrin and its amino (N)-terminal lobe at the physiologically relevant pHs 7.4 and 5.0, we have combined ultraviolet circular dichroism (CD) spectroscopy with transient-electric birefringence (TEB) measurements. No significant changes are found in the protein's secondary structure under the different conditions studied. The tertiary structure as monitored by near-UV CD is affected by iron binding, but does not change upon decrease in pH. In contrast, TEB results indicate dramatic changes in the dynamic structure of transferrin both upon binding of iron and decrease of pH. In apotransferrin freedom of movement is found for the lobes with respect to each other, and for the domains within the lobes. The interlobal flexibility is considerably enhanced at the lower pH. Holotransferrin is found to behave as a rigid molecule.

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).

Further characterization of structural and electric properties of non-spherical alpha-crystallin.

Recently we have shown that the population of native alpha-crystallin, isolated using size-exclusion chromatography from eye lenses of calves, is multimodal. Most of the protein probably possesses an almost spherical appearance, but at least one of the other modes represents more extended, ellipsoidally and/or cylindrically shaped molecules [Van Haeringen, B., Eden, D., Van den Bogaerde, M.R., Van Grondelle, R. & Bloemendal, M. (1992) Eur. J. Biochem. 210, 211-216]. In the present study, we characterize various subpools of a single alpha-crystallin size-exclusion chromatography elution peak by means of transient-electric-birefringence measurements, ultraviolet linear-dichroism spectroscopy and analytical fast protein liquid chromatography. It is concluded that the fractions have a well-defined stable mass and are not in reversible equilibrium with each other. All pools appear to be composed of at least two types of differently shaped molecules. The hydrodynamic dimensions and electric properties of the different alpha-crystallin species are characterized. The non-spherical alpha-crystallin is found to be optically and electrically more anisotropic, and to contain a larger permanent electric dipole moment than the spherical form. A model for the composition of the alpha-crystallin pool is presented.

Alpha-crystallin exists in a non-spherical form. A study on the rotational properties of native and reconstituted alpha-crystallin.

Native alpha-crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient-electric-birefringence measurements and ultraviolet linear-dichroism spectroscopy. These techniques were also performed on 6-M-urea-dissociated and reconstituted alpha-crystallin. Transient-electric-birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non-spherical occurrences of alpha-crystallin in more detail. Although not distinguishable with size-exclusion chromatography, we could identify at least two different classes of both native and reconstituted alpha-crystallin, from which at least one consists of non-spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the alpha-crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three-layer model for alpha-crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711-724].

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.

Polarized fluorescence measurements on ordered photosynthetic antenna complexes: Chlorosomes of Chloroflexus aurantiacus and B800-B850 antenna complexes of Rhodobacter sphaeroides.

We have used a new and relatively easy approach to study the pigment-organization in chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus and in B800-850 antenna complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides. These particles were embedded in compressed and uncompressed gels and the polarized fluorescence was determined in a 90 degrees setup. Assuming both a rotational symmetric distribution of the particles in the gel and of the transition dipole moments in the particles, the order parameters and , describing the orientation of the symmetry axis of the particles with respect to the direction of gel expansion can be determined. Moreover, the direction parameters, describing the orientation of the absorption and emission dipole moments with respect to the symmetry axis of the particles can be obtained.The value of is essential for quantitative interpretation of linear dichroism measurements and usually it is estimated from theoretical approaches, which may lead to incorrect results. For the rod-like chlorosomes the value of appears to be the same as predicted by the theoretical approach of Ganago, A. O., M. V. Fok, I. A. Abdourakhmanov, A. A. Solov'ev, and Yu. E. Erokhin (1980. Mol. Biol. [Mosc.]. 14:381-389). The agreement with linear dichroism results, analyzed with this theoretical approach shows that the transition dipole moments are indeed in good approximation distributed in a rotationally symmetric way around the long axis of the chlorosomes. Moreover, it appears those BChl c molecules, which fluoresce, are oriented in the same way with respect to the symmetry axis as the rest of these pigments, with the dipole moments close to parallel to the long axis.The B800-850 complexes appear to orient like discs, whereas the transition dipoles of the BChl a 800- and 850-nm bands are oriented almost perpendicular to the symmetry axis. These findings are in agreement with the minimal model for these complexes proposed by Kramer, H. J. M., R. van Grondelle, C. N. Hunter, W. H. J. Westerhuis, and J. Amesz (1984. Biochim. Biophys. Acta. 156-165).The amount of orientation of the particles appears to vary for different gels and it is lower than predicted by the theory of Ganago et al., showing that application of their approach for these particles leads to incorrect interpretations.The approach that is used in this study allows determination of orientations of those dipole moments, which transfer their excitation energy to the fluorescing species, in contrast to linear dichroism measurements, where the orientations of all absorbing dipole moments are studied. For the polarized fluorescence measurements, the amount of orientation of the particles is determined experimentally, whereas for linear dichroism this amount has to be estimated from theoretical models. The value of that can be determined from the fluorescence measurements can, however, also be used for a quantitative interpretation of the linear dichroism results.

A refined calculation of the solution dimensions of the complex between gene 32 protein and single stranded DNA based on estimates of the bending persistence length.

The rotation diffusion coefficient of a complex of GP32, the single stranded DNA binding protein of the bacteriophage T4, with a single stranded DNA fragment with about 270 bases was determined to obtain further information on the flexibility of this particle. The rotation diffusion of these molecules is used as a sensitive measure of the flexibility of different DNA protein complexes. Using the theory of Hagerman and Zimm (Biopolymers 20, 1481 (1981)) and assuming a bending persistence length of about 35 nanometer it can be shown that the axial increment for GP32 complexes with single stranded DNA is close to 0.5 nm per base. The value for the bending persistence length is in agreement with values found for much larger DNA protein complexes using light scattering experiments. This value for the persistence length also implies that the complex is thin. The radius is estimated to be around 1.7 nm, which shows a moderate degree of hydration. With this set of parameters we can describe all the hydrodynamic experiments on GP32 complexes from 76 to more than 7000 bases obtained using electric birefringence, quasi-elastic light scattering and sedimentation experiments performed in our group over the last few years.