Dietrich Zirwer

Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein.

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90–232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90–232 showed an infrared absorbance spectrum typical of proteins with a predominant α-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3–2 m guanidine hydrochloride), the secondary structure of SHaPrP90–232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel β-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in α-helical structures and the increase in β-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No β-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90–232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as “critical oligomers.” The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90–232. A new model for the structural transition and aggregation process of the prion protein is described.

Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions

Abstract. The conformational transitions of bovine β-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of β-lactoglobulin A from the native to the highly helical state is clearly demonstrated.

COMPACTNESS OF PROTEIN MOLTEN GLOBULES : TEMPERATURE-INDUCED STRUCTURAL CHANGES OF THE APOMYOGLOBIN FOLDING INTERMEDIATE

Apomyoglobin undergoes a two-step unfolding transition when the pH is lowered from 6 to 2. The partly folded intermediate (1) state at pH 4 and low ionic strength has properties of a molten globule. We have studied structural features of this state, its compactness, content of secondary structure, and specific packing of aromatic side chains, using dynamic light scattering, and small-angle X-ray scattering and far- and near-ultraviolet circular dichroism spectroscopy. Particular attention was paid to temperature-dependent structural changes. The results are discussed with reference to the native-like (N) state and the highly unfolded (U) state. It turned out that the I-state is most compact near 30°C, having a Stokes radius 20% larger and a radius of gyration 30% larger than those of the N-state. Both cooling and heating relative to 30°C led to an expansion of the molecule, but the structural changes at low and high temperatures were of a different kind. At temperatures above 40°C non co-operative melting of structural elements was observed, while the secondary structure was essentially retained on cooling. The results are discussed in context with theoretical predictions of the compactness and the stability of apomyoglobin by Alonso et al. [Alonso, D. O. V., Dill, K, A., and Stigler, D. (1991) Biopolymers 31:1631–1649]. Comparing the I-state of apomyoglobin with the molten globules of α-lactalbumin and cytochrome c, we found that the compactness of the molten globule states of the three proteins decreases in the order α-lactalbumin > apocytochrome c > apomyoglobin. While α-lactalbumin and cytochrome c are rather homogeneously expanded, apomyoglobin exhibits a non uniform expansion, since two structural domains could clearly be detected by small-angle X-ray scattering.

Quasielastic light scattering from human α-lactalbumin: comparison of molecular dimensions in native and ‘molten globule’ states

Abstract Quasielastic light scattering has been applied to compare the linear dimensions of human α-lactalbumin molecules in the native ‘molten globule’ and unfolded states. The translational diffusion coefficients of the protein have been measured at neutral and acid pH as well as in 6 m guanidine hydrochloride. Temperature dependence of diffusion coefficients for Ca2+-free protein at neutral and acid pH have also been obtained. After correction for the protein association, it is shown that the effective linear dimensions of protein molecules increase by about 10% in the ‘molten globule’ state (i.e. at acid pH or high temperature) as compared with the native state. This increase in linear dimensions is much smaller than the increase in the unfolded state (∼40%

Conversion of yeast phosphoglycerate kinase into amyloid-like structure

Yeast phosphoglycerate kinase is a structurally well‐characterized enzyme consisting of 415 amino acids without disulfide bonds. Anion‐induced refolding from its acid‐unfolded state gives rise to the formation of worm‐like amyloid fibrils with a persistence length of 73 nm. Electron microscopy and small‐angle X‐ray scattering data indicate that the fibrils have an elliptical cross‐section with dimensions of 10.2 nm × 5.1 nm. About half of all amino acids are organized in form of cross‐β structure which gives rise to typical infrared spectra, X‐ray diffraction and yellow‐green birefringence after Congo red staining. The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X‐ray scattering, was found to be strongly dependent on protein concentration. The infrared data indicate that the formation of cross‐β structure practically comes to an end already after some hours, whereas the length‐growth of the amyloid fibrils, monitored by small‐angle X‐ray scattering, was not yet completed after 1,300 hours. Proteins 2000;39:204–211.

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

Abstract 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 Glockner. 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 Glockner 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.

Shape, symmetry, hydration and secondary structure of the legumin from Vicia faba in solution

The following physical parameters of the legumin from Vicia faba were determined by means of small-angle X-ray scattering, quasi-elastic light scattering and circular dichroism: molar mass, M = 3.5 X 10(5) g/mol; radius of gyration, Rg = 4.45 nm; maximum dimension, L = 13 nm; translational diffusion coefficient, D0(20),w = 3.38 X 10(-7) cm2 X s-1; alpha-helix content about 15%; content of beta-sheets 10%; dihedral point group symmetry of the molecule 32.

Application of dynamic light scattering to studies of protein folding kinetics

The applicability of dynamic light scattering to studies of the kinetics of unfolding and refolding reactions of proteins is discussed and demonstrated experimentally. The experimental set-up and the data acquisition and data evaluation schemes that have been optimized for kinetic experiments are described. The relationship of the signal-to-noise ratio to the minimum data acquisition time that is needed to obtain results of sufficiently high precision is discussed. It turns out that the attainable time resolution is of the order of a few seconds for proteins with molar masses of about 50,000 g · mol−1 and concentrations of 1 g · l−1. Thus, DLS is too slow to follow conformational changes in the subsecond region, but it is useful for studies of unfolding-refolding reactions of proteins that proceed with time constants in the range of seconds or minutes. This is demonstrated by investigations of the kinetics of the cold denaturation of 3-phosphoglycerate kinase from yeast.

Auto-oxidation-induced fusion of lipid vesicles.

Spontaneous size changes of small unilamellar vesicles with initial mean diameters of 25 nm measured by quasi-elastic light scattering (QELS) and electron microscopy are reported. After the size conversion the vesicles have mean diameters of about 70 nm and are of the unilamellar and multilamellar type. The fact that auto-oxidation initiates this process is established by the comparison of the results for vesicles which differ only in the degree of auto-oxidation. The role of phosphatidylcholine hydroperoxides as fusogens is discussed.

Physical and conformational properties of staphylokinase in solution

The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy. Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm. The sedimentation coefficient is 1.71 S. These physical parameters indicate that the shape of staphylokinase is very elongated. The protein molecule consists of two folded domains of similar size. The mean distance of the centres of gravity of the domains is 3.7 nm. The mutual positions of the two domains are variable in solution. Thus, the molecule is shaped like a flexible dumbbell. About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in beta-sheets and 20% form turns.