Hilde Damaschun

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.

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.

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.

Chloroplast coupling factor CF1 in solution Small-angle X-ray scattering and circular dichroism measurements

Recently a proton translocating protein complex was shown to catalyze the photosynthetic ATP synthesis of chloroplasts when the thylakoid membranes are stressed by a transmembrane proton gradient as proposed [l-3] . This complex involves at least the peripheral membrane protein coupling factor CFI being the actual ATPsynthetase and a nonidentified transmembrane component designated HFo [3,4] . From this complex only the coupling factor could be purified to homogeneity. This protein with a molecular weight of about 320 000 [5] could be resolved into five subunit classes (a, /3, y, 6, E) by sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. They have mol. wt 59 000,55 000,37 000, 2 1 000 and 16 000, respectively [6-81. The relative staining intensities of the subunit bands from the gels and crosslinking experiments gave evidence for one molecule of CF, to be composed of ICY, 20, ly, 16 and 2~ subunits [9-l 11. Though conformational changes of CF1 subunits have been detected during the process of photophosphorylation [ 121 only little is known on the quaternary structure of the enzyme. Electron microscopic data have shown CFI to be spherical in shape, but there exist contradictory results with respect to the diameter of this particle. Previously the diameter of the membrane-bound as well as of the solubilized CFI was found to be 150 A in freezeetched preparations and 90 A in negatively-stained material [ 131 . Thus electron-optical investigations of the quater-

Ribonuclease T1 has different dimensions in the thermally and chemically denatured states: a dynamic light scattering study.

© 1997 Federation of European Biochemical Societies.

Solvent dependence of dimensions of unfolded protein chains

The radii of gyration of unfolded apo-cytochrome C at pH 2.3 have been determined in three conditions: (i) 20 mM sodium phosphate buffer; (ii) 0.25 M NaCl; and (iii) 6.65 M GuHCl by small-angle X-ray scattering, and (iii) from translational diffusion coefficients measured by dynamic light scattering. The radius of gyration of the unfolded protein chain depends remarkably on the quality of the solvent, decreasing in the order 20 mM sodium phosphate greater than 6.65 M GuHCl greater than 0.25 M NaCl. The value of the radius of gyration in 0.25 M NaCl and also the value estimated for infinite ionic strength are close to the value predicted theoretically for the theta-point. This means that water in the absence of electrostatic interactions is a poor solvent for an unfolded protein while 6.65 M GuHCl is a better solvent.

Marine invertebrate sperm-specific histones and histone-DNA interactions: circular dichroism and ultraviolet spectroscopy studies

Abstract Circular dichroism (c.d.) and turbidity measurements have been performed to study the structural behaviour of the marine invertebrate sperm-specific histones H1, H2B and of the mollusc sperm-specific protein S2 and their dependence on ionic strength as well as the formation of complexes of these proteins with DNA. Sea urchin sperm histones H1 from Strongylocentrotus intermedius or Strongylocentrotus droebachiensis attain, at 2 m NaCl, a higher helicity than histones H1 from the sperm of the bivalve mollusc Chlamis islandicus or from calf thymus. This extra helical part of the sea urchin sperm histones H1 is rapidly digested by trypsin. The complexes of the marine invertebrate H1 and S2 proteins with DNA are characterized by c.d. spectra with strong negative ellipticities corresponding to the (-PSI)-type c.d. spectra of calf thymus H1-DNA complexes. The arginine content of the histones correlates with the ionic strength at which the formation of the protein—DNA complexes begins. The higher the arginine content the higher is the ionic strength at which complex formation sets in once ionic strength is reduced. The results of the investigations on H2B-DNA complexes point to a possible involvement of H2B from sea urchin sperm in the condensation of sea urchin sperm chromatin.

Small-angle X-ray scattering studies of the structure of nucleosome core histone complexes in solution

Abstract The nucleosome core histone complex in solution at 2 M NaCl and pH 7 has a radius of gyration Rs, of 3.48 nm and a maximum dimension, L, of 12 nm. Its shape is disc-like with a mean thickness of 3 nm. The radius of gyration determined by us is of the same value as the radius of gyration of the complex in intact core particles (Braddock) et al., Biopolymers 1981, 20, 327). Thus, we conclude that the basic histone tails of the protein complex project about 2 nm from its central part.