Papers by Michael Bloemendal
The Use of Fast Protein Liquid (Size Exclusion) Chromatography for the Fractionation of Crystallins and the Study of β-Crystallin Aggregation
Journal of Liquid Chromatography, 1993
ABSTRACT
A structural study of bovine lens alpha-crystallin and its subunits by absorption and linear dichroism spectroscopy
European Journal of Biochemistry, 1989
The structure of the bovine alpha-crystallin aggregate and its reaggregated isolated subunits has... more The structure of the bovine alpha-crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250-350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in alpha-crystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of alpha-crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.
European Journal of Biochemistry, 1993
Recently we have shown that the population of native α‐crystallin, isolated using size‐exclusion ... more Recently we have shown that the population of native α‐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 α‐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 prop...
α‐Crystallin exists in a non‐spherical form
European Journal of Biochemistry, 1992
Native α‐crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characteri... more Native α‐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 α‐crystallin. Transient‐electric‐birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non‐spherical occurrences of α‐crystallin in more detail. Although not distinguishable with size‐exclusion chromatography, we could identify at least two different classes of both native and reconstituted α‐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 α‐crystallin exists in a more extended quaternary structure. The results are difficult to explain with a...
Non-random conformation of a mouse IgG2a monoclonal antibody at low pH
European Journal of Biochemistry, 1991
The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was inve... more 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.
Linear-dichroism spectroscopy for the study of structural properties of proteins
Molecular Biology Reports, 1993
This review gives an experiment directed survey of the application of linear-dichroism (LD) spect... more This review gives an experiment directed survey of the application of linear-dichroism (LD) spectroscopy to the study of proteins. LD spectroscopy is a relatively simple technique that provides information on the orientation of chromophores in molecules, on molecular characteristics such as shape, size and electronic properties, and on binding parameters in molecular complexes. Since LD is only observed when the molecules are non-randomly oriented in the sample, particular attention is paid to various orientation techniques, viz. in electric and flow fields, in polymer films and gels, and by light induction (photoselection). Examples are given on bacteriorhodopsin and retinals, chlorosomes, lens crystallins, aspartate aminotransferase, and the interaction of gene32- and recA-protein with DNA.
Enthalpic interaction coefficients of formamides dissolved in N,N-dimethylformamide
Journal of Solution Chemistry, 1986
ABSTRACT
Correlation of solute-solute interaction enthalpies in dimethylformamide by group additivity
Journal of Solution Chemistry, 1987
... Group Additivity Michael Bloemendal, 1'2 and Gus Somsen 1'3 Received Decemb... more ... Group Additivity Michael Bloemendal, 1'2 and Gus Somsen 1'3 Received December 9, 1986 The Savage and Wood group additivity method has been applied to enthal-pic pair interaction coefficients of more than 30 solutes dissolved in N,N-dimethylformamide. ...
The random contact point model for pair interaction coefficients of unlike solute molecules
Journal of Solution Chemistry, 1989
ABSTRACT
Characterization of multiple oligomeric vimentin intermediate filament units by transient electric birefringence measurements
Journal of Molecular Biology, 1994
In this work we have studied the structure of soluble vimentin units from which intermediate fila... more In this work we have studied the structure of soluble vimentin units from which intermediate filaments (IFs) are built. Several oligomeric forms have been presented in the literature as IF "building blocks", but there is still no agreement on this matter. By comparing our data with various models as proposed in the literature we can favour certain models and reject others. Transient electric birefringence (TEB) measurements were performed from which information is obtained concerning electric and hydrodynamic properties of the particles under investigation. TEB decay analysis at pH 6.8 after 70 microseconds pulses (at 20 degrees C in aqueous solution) yielded three decay times: 1.1(+/- 0.3) microseconds, 4.0(+/- 1.0) microseconds and 20.0(+/- 5.0) microseconds, with amplitudes of 45% to 60%, 30% to 45%, and less than 10%, respectively. At pH 8.5 after 70 microseconds pulses, more than 90% of the TEB signal with the second decay time is obtained, while the remainder had a decay time of 15.0(+/- 4.0) microseconds. Only when the pulse duration was decreased, the fast decay time around 1 microsecond was observed, suggesting that only a minor fraction of the particles at this pH value causes such a short decay time. At both pH values, the TEB measurements indicated that, at least in part, the molecules are oriented by a permanent dipole moment. It will be shown that the shortest decay time originates from bent or flexible dimers, and the second decay time from particles with a length of 54 to 65 nm containing, at least in part, a relatively large overall dipole moment. The longest decay time is probably due to larger aggregates. These results are consistent with a model in which single dimers, antiparallel staggered tetramers and hexamers coexist. Alternatively, but less likely on the basis of literature data, a model of parallel in-register tetramers with a considerable length contribution of the head groups would fit our research.
Journal of Molecular Biology, 1990
The structures of the two very closely related proteins, bovine ~II-and TIVa-crystallin have been... more The structures of the two very closely related proteins, bovine ~II-and TIVa-crystallin have been studied by means of near-ultra-violet linear dichroism spectroscopy on squeezed polyacrylamide gel systems. The crystaltin spectra are discussed in terms of the spectra of the aromatic chromophores present in these proteins and provide detailed information on the average orientation of these residues in the proteins. A comparison of our results with information based on crystallographic X-ray experiments shows excellent agreement, reflecting even some of the minor differences between the two proteins studied. Since linear dichroism measurements as performed here take a few days only, and can be done on most aqueous protein solutions, linear dichroism spectroscopy may give a valuable contribution to structural studies on proteins.
Molar refractivities of tetra-n-alkylammonium salts and ions
Journal of Chemical & Engineering Data, 1988
ABSTRACT
Acid-Induced Structural Changes of a Mouse IgG 2a Monoclonal Antibody (MN12) Studied by Transient Electric Birefringence Measurement
Journal of Biomolecular Structure and Dynamics, 1992
Acid-induced structural changes of a mouse IgG2a monoclonal antibody (MN12) as indicated by Jisko... more 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.
Journal of Biological Chemistry, 1995
FEBS Letters, 1994
Fourier transform infrared spectroscopy has been used to study the solution structure and thermal... more Fourier transform infrared spectroscopy has been used to study the solution structure and thermal stability of the extracellular fragment of human transferrin receptor (tfRt) at extracellular and endosomal pH. At extracellular pH t!Rt is composed of 56% a-helix, 19%B-sheet and 14% turns. Upon acidification to endosomal pH the a-helical content of the protein is reduced and the B-sheet content increased by nearly 10%. At extracellular pH, the midpoint temperature of thermal denaturation (T,) for the loss of secondary and tertiary structure, and the formation of aggregated structures, is 7 1 "C. At endosomal pH this temperature is reduced by 2 15°C. The apparent entropies of thermal denaturation indicate that the native structure of tfRt at endosomal pH is far more flexible than at extracellular pH.
FEBS Letters, 1992
A selective tyrosine fluorescence quenching is found on interaction of vimentin with poly(dT) and... more A selective tyrosine fluorescence quenching is found on interaction of vimentin with poly(dT) and poly(rA). However, addition of poly(dA) does not result in tyrosine quenching. The number of nucleotides covered by vimentin upon binding(n) of poly(dT) (50 ± 4) appeared to be approximately the same as for poly(rA) (44 ± 4), while the apparent binding constant (K app) of the latter is slightly larger (5.0 ± 2.0 × 107 M−1·cm−1 vs. 2.5 ± 0.5 × 107 M−1·cm−1). The finding that there exists a specific strong interaction between vimentin and nucleic acids could help in the search for cellular functions of intermediate filament proteins.
FEBS Letters, 1995
The low ionic strength structures of the type III intermediate filament (IF) proteins desmin and ... more The low ionic strength structures of the type III intermediate filament (IF) proteins desmin and glial fibrillary acidic protein (GFAP) have been studied by transient electric birefringence measurements. Flexible dimers with a length of around 45 nm, particles with a length of 68 -+ 6 nm (presumably tetramers and hexamers) and larger aggregates of 108 -+ 19 nm are found. GFAP has an increased tendency to aggregate upon lowering of the pH. The aggregation state of desmin does not change in the pH range studied. The results are compared with previous results on vimentin.
Native exists in a non-spherical form
Experimental Eye Research, 1992
Experimental Eye Research, 1995
The prevailing water-soluble proteins in the lens are the so-called crystallins. In mammals these... more The prevailing water-soluble proteins in the lens are the so-called crystallins. In mammals these occur mainly in three classes: ~ (MW about 800 kDa), /? (MW 50-300kDa) and 7 (MW about 20kDa) . Based on their chromatographic behaviour the/%crystallins are divided into two groups •H(igh) and flL(ow). In some cases the latter is further
Biophysical Journal, 1994
We have developed a straightforward method to separate linear-dichroism and birefringence contrib... more 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- f3-D-maltoside and n-octyl-3-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,
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Papers by Michael Bloemendal