Effect of environmental conditions on aggregation and fibril formation of barstar.

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-beta structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state-->fibril type I-->fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.

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 x 5.1 nm. About half of all amino acids are organized in form of cross-beta 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-beta 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 can adopt totally different folded conformations.

The three-dimensional structure of a protein is determined by interactions between its amino acids and by interactions of the amino acids with molecules of the environment. The great influence of the latter interactions is demonstrated for the enzyme phosphoglycerate kinase from yeast (PGK). In the native state, PGK is a compact, bilobal molecule; 35% and 13% of its amino acids are organised in the form of alpha-helices and beta-sheets, respectively. The molecules unfold at acidic pH and low ionic strength forming random-walk structures with a persistence length of 3 nm. More than 90% of the amino acid residues of the ensemble have phi,psi-angles corresponding to those of a straight beta-chain. Upon addition of 50% (v/v) trifluoroethanol to the acid-unfolded protein, the entire molecule is transformed into a rod-like, flexible alpha-helix. Addition of anions, such as chloride or trichloroacetate, to the acid-unfolded protein leads to the formation of amyloid-like fibres over a period of many hours when the anion concentration exceeds a critical limit. Half of the amino acid residues are then organised in beta-sheets. Both of the non-natively folded states of PGK contain more regular secondary structure than the native one. The misfolding starts in both cases from the acid-unfolded state, in which the molecules are essentially more expanded than in other denatured states, e.g. those effected by temperature or guanidine hydrochloride.

Denatured states of yeast phosphoglycerate kinase.

Structures of proteins in unfolded states have important implications for the protein folding problem and for the translocation of polypeptide chains. Acid-denatured, cold-denatured, and 6 M guanidine hydrochloride (GuHCl) denatured yeast phosphoglycerate kinase (PGK) are ensembles of flexible unfolded molecules with rapidly interconverting structures of the individual polypeptide chains. They differ, however, in their physical properties, such as in coil size and in stiffness over a short distance along the chain. These properties of polypeptide chains can be described well by persistence statistics. A solution containing 0.7 M GuHCl at 4.5 degrees C is nearly a Theta-solvent for PGK. By contrast, 6 M GuHCl is a good solvent for PGK. Acid-denatured PGK at low ionic strength has the most expanded and stiffest chains. The conformation of heat-denatured PGK should be more compact than that of random walk chains at the Theta-point, as can be inferred from measurements on other proteins. Investigations of heat-denatured PGK by scattering methods are unfeasible due to aggregation of the protein. The persistence length as a measure of chain stiffness varies between a = 1.74 nm for cold-denatured PGK and a = 3.0 nm for acid-denatured PGK. The distribution functions of the gyration radii were calculated from the X-ray scattering data for all unfolded states and compared with the radius of gyration of the natively folded molecule.

Conformation of thermally denatured RNase T1 with intact disulfide bonds: a study by small-angle X-ray scattering.

Small-angle X-ray scattering of RNase T1 with intact disulfide bonds was measured at 20 degrees and 60 degrees C in order to get insight into the structural changes of the protein caused by thermal denaturation. The radius of gyration increases from R(G)= 1.43 nm to R(G) = 2.21 nm. The conformations of the molecules at 60 degrees C are similar to those of ring-shaped random walk chains. However, the molecules are more compact than one would expect under theta conditions due to attractive interactions between the chain segments. The volume needed for free rotation of the thermally unfolded protein molecules about any axis in solution is five times greater than in the native state whereas the hydrodynamic effective volume is increasing only two times.

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

Ribonuclease T1 can be unfolded and refolded without forming noticeable amounts of aggregates allowing to characterise the dimensions of a protein in different denatured states in terms of the Stokes radius RS. Upon thermal unfolding RS increases from 1.74 nm at 20 degrees C to 2.14 nm at 60 degrees C. By contrast, RS = 2.40 nm was obtained at 5.3 M guanidinium chloride (GuHCl) and 20 degrees C. Heating from 20 degrees C to 70 degrees C in the presence of 5.3 M GuHCl led to a 5% decrease in RS.

Prothymosin alpha: a biologically active protein with random coil conformation.

Prothymosin is an acidic protein with an unusual amino acid composition. Though its exact function is not yet known, its high evolutionary conservation and wide tissue distribution suggest an essential biological role. Its physical state, which is controversially discussed in previous publications, was investigated using small-angle X-ray scattering, dynamic light scattering, mass spectrometry, and circular dichroism (CD). Our results unequivocally demonstrate that prothymosin is a monomer under physiological conditions. The protein adopts a random coillike conformation but exhibits persistence of direction and curvature. No regular secondary structure is detectable by CD. The Stokes radius, Rs = 3.07 nm, and the radius of gyration, RG = 4.76 nm, are 1.77 and 3.42 times larger, respectively, than those expected for a compactly folded protein consisting of 109 amino acid residues. A remarkable amount of secondary structure is formed only in the presence of trifluoroethanol at low pH. The finding that a biologically active protein molecule with 109 amino acid residues adopts a random coil conformation under physiological conditions raises the question whether this is a rare or a hitherto-overlooked but widespread phenomenon in the field of macromolecular polypeptides.

The thermostability of natural variants of bacterial plasminogen-activator staphylokinase.

Three natural variants (wild-type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase) of the bacterial plasminogen-activator staphylokinase, a 136-amino-acid protein secreted by certain Staphylococcus aureus strains, have been characterized. These variants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogen-activating capacity and conformation in solution, as revealed by fluorescence spectroscopy, dynamic light scattering and circular dichroism. However, the thermostability of these variants is significantly different. At 70 degrees C and 0.5 mg protein/ml, irreversible inactivation occurred with apparent half-life (t1/2) values 0.54 +/- 0.13, 0.81 +/- 0.20 and 3.7 +/- 0.7 h (mean +/- SEM) for wild-type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase, respectively, with corresponding values at 0.08 mg/ml of 5.3 +/- 1.4 h and 11 +/- 2.0 h for wild-type staphylokinase and [R36G, R43H]staphylokinase, respectively. Dynamic light-scattering measurements indicated that inactivation was associated with protein aggregation, which precluded accurate determination of transition temperatures and enthalpies of unfolding. 0.08-0.34 mg/ml [G34S, R36G, R43H]staphylokinase, however, did not aggregate at 70 degrees C but underwent unfolding as revealed by a 20% increase in the Stokes' radius and a 30% decrease in circular dichroism. The unfolding was reversible upon cooling and was associated with full recovery of functional activity. Thus, these natural variants of staphylokinase have a different sensitivity to thermal inactivation, that is mediated by reversible unfolding of the protein and concentration-dependent irreversible aggregation. [G34S, R36G, R43H]staphylokinase, the most resistant natural variant, has a stability approaching the minimal requirements for pasteurization, which would facilitate its development for clinical use.

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 (I) 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 degrees 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 degrees 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 degrees 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 Stigter, D. (1991) Biopolymers 31:1631-1649]. Comparing the I-state of apomyoglobin with the molten globules of alpha-lactalbumin and cytochrome c, we found that the compactness of the molten globule states of the three proteins decreases in the order alpha-lactalbumin > apocytochrome c > apomyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold denaturation-induced conformational changes in phosphoglycerate kinase from yeast.

The temperature-dependent conformational equilibrium of 3-phosphoglycerate kinase has been studied in the temperature range from 1 to 30 degrees C by means of dynamic light scattering, small-angle X-ray scattering, differential scanning calorimetry, circular dichroism spectroscopy, and fluorescence spectroscopy. At 28 degrees C and in the presence of 0.7 M guanidine hydrochloride (GuHCl), the radius of gyration (RG) and the Stokes radius (RS) are 2.44 and 3.09 nm, respectively. Decreasing the temperature effects unfolding of the molecule, a process that involves two stages. The two stages correspond to the successive unfolding of the N-terminal and C-terminal domains. The peak maxima of the excess heat capacity, determined from differential calorimetric scans, extrapolated to 0 scan rate, are positioned at 16.5 degrees C for the N-terminal domain and at 6.3 degrees C for the C-terminal domain. At 4.5 degrees C, the radius of gyration and the Stokes radius increase to 7.8 and 4.8 nm, respectively. The persistence length and the length of the statistical chain segment of the unfolded polypeptide chain are 1.74 and 3.48 nm, corresponding to five and ten amino acids, respectively. At 1 degrees C, the dimensions of the unfolded chain nearly agree with the predicted dimensions under theta conditions. Thus, the conformational changes upon cold denaturation can be described by a transition from a compactly folded molecule to a random coil. The conformation-dependent ratio rho = RGRS-1 increases from rho = 0.79 to rho = 1.63. The volume of the unfolded chain is 30 times larger than that of the folded chain in the native state.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold denaturation of yeast phosphoglycerate kinase: kinetics of changes in secondary structure and compactness on unfolding and refolding.

Under mildly destabilizing conditions (0.7 M GuHCl), phosphoglycerate kinase from yeast undergoes a reversible two-step equilibrium unfolding transition when the temperature is lowered from 30 to 1 degree C (Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278). The kinetics of the changes in compactness and secondary structure have been studied by means of dynamic light scattering and far-UV circular dichroism, respectively. It turned out that unfolding and refolding after an appropriate temperature jump (T-jump) was performed proceeded in substantially different ways. After a T-jump from 30 to 1 degree C, a multiphasic unfolding behavior was observed, reflecting the independent unfolding of the N-terminal and C-terminal domains with time constants of about 7 and 45 min, respectively. A remarkable feature of the unfolding process is the simultaneous change of compactness and secondary structure. Refolding after a T-jump from 1 degree C to higher temperatures occurs in two stages. At the first stage an appreciable amount of secondary structure is formed rapidly within the dead time of the T-jump, while the overall dimensions of the polypeptide chain remain essentially unchanged. Thus, an extended folding intermediate is formed at an early stage of folding. Further information of secondary structure proceeds slowly within a time range of minutes in parallel with the increase of compactness. At 30 degrees C, both domains refold simultaneously, while at 15 degrees C, independent folding can be observed. These findings are discussed with respect to predictions of existing models of folding.

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.

Streptokinase is a flexible multi-domain protein.

The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.

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.

Acid denatured apo-cytochrome c is a random coil: evidence from small-angle X-ray scattering and dynamic light scattering.

The conformation of a denatured protein has been investigated, since the experimental data on the structure of denatured proteins have been incomplete until now. The Stokes' radius Rs and the radius of gyration Rg of apo-cytochrome c at pH 2.3 have been determined by dynamic light scattering and small-angle X-ray scattering, respectively. The values of these structure parameters, extrapolated to zero protein concentration, are Rs = 3.0 nm and Rg = 4.6 nm. The ratio Rg.Rs-1 is a sensitive indicator of the molecular conformation. The ratio of 1.55 obtained by us is typical for a random-coil polymer. The persistence length--the characteristic of the molecular flexibility--was determined to be a = 1.81 nm. From this results the root-mean-square average end-to-end distance of the molecules [h2] 1/2 = 11.2 nm and the characteristic ratio [h2]/npl2p = 8.43, where np = 104 is the number of amino acid residues and lp the distance between C alpha-atoms. We obtained a second virial coefficient A2 = 8.2.10(-3) mol cm3 g-2. The experimentally determined structure parameters are in approximate agreement with those predicted by Flory and others for an unperturbed, randomly coiled polypeptide. The expansion factor lies between 1.1 and 1.2. In conclusion, we have shown that apo-cytochrome c at pH 2.3 and at low concentrations has the conformation of a perturbed random coil with repulsive potentials between the chain segments.

Effect of platinum(II) chemotherapeutic agents on properties of DNA liquid crystals.

We have investigated the X-ray and optical properties (CD spectra and polarization microscopy) of liquid-crystalline phases and dispersions formed on pretreatment of low molecular weight DNA with the platinum(II) coordination complexes, cis-diammine-dichloroplatinum(II) (DDP), 2,2'-bipyridinedichloroplatinum(II) (1) and 2,2'-bipyridineethylenediammineplatinum(II) (2). It is demonstrated that the platination of DNA leads to the ordering of neighbouring molecules of DNA in liquid-crystalline phases being diminished. The intense bands observed in the CD spectra of liquid-crystalline dispersions prepared from DNA pretreated with 1 or 2 can be used to determine the orientation of the latter compounds with respect to the helical axis of the DNA and to detect distortions in the secondary structure of DNA. The possible causes of the appearance of the intense bands in the CD spectra of liquid-crystalline phases and alterations in the manner of packing of the molecules of DNA within them are discussed.

[2 types of temperature transitions in liquid crystals formed from poly(I).poly(C) molecules]. / Dva tipa temperaturnykh perekhodov zhidkikh kristallov, sformirovannykh iz molekul poli(I).poli(C).

The small-angle X-ray scattering curves, CD spectra and textures of the liquid-crystalline phase formed from poly(I).poly(C) molecules in a water-salt solutions containing poly(ethylene glycol) at different temperatures were obtained. It was found that the heating of poly(1).poly(C) liquid-crystalline phase is accompanied by two types of transitions, the first one--a "cholesteric----"compensated" structure----cholesteric", the second--a "cholesteric----isotropic state" transition. The latter transition takes place at a temperature that corresponds to that of the separation of chains of the double-stranded poly(I).poly(C) molecule.

X-ray scattering evidence that calf thymus DNA in solution is a double helix and not a warped zipper.

Isotropic X-ray scattering experiments with calf thymus DNA in solution under B-form conditions were used to differentiate between the double helical and the side by side structure models. By comparison of experimental and theoretical scattering curves calculated from the atomic coordinates of the molecule models, two sterically refined SBS models can be excluded for calf thymus DNA. The structural basis of the differences between the experimental scattering curves and the theoretical curves for the double helix on the one hand and for the two SBS models on the other, is interpreted using high resolution electron distance distribution functions of the models.

How many base-pairs per turn does DNA have in solution and in chromatin? An answer from wide-angle X-ray scattering.

Experimental excess wide-angle X-ray scattering curves from DNA in solution, from Na-DNA crystallites in mother-liquor, from mononucleosomes in solution and from nucleosome core histone complexes are compared with each other and with calculated excess scattering curves of DNA with variable number of base-pairs per turn. The DNA in the Na-DNA crystallites and in the mononucleosomes has on average the canonical B-conformation which is well-known from fibre-diagrams with 10 base-pairs per turn and a rise of 0.34 nm per base-pair. The averaged structure of DNA in solution differs from this conformation. The model which fits best this structure has 10.8 base-pairs per turn and a rise of 0.34 nm per base-pair.