Polymerization of proteins into amyloid protofibrils shares common critical oligomeric states but differs in the mechanisms of their formation.

Amyloid protofibril formation of phosphoglycerate kinase (PGK) and Syrian hamster prion protein (SHaPrP(90-232)) were investigated by static and dynamic light scattering, size exclusion chromatography and electron microscopy. Changes in secondary structure were monitored by Fourier transform infrared spectroscopy and by circular dichroism. Protofibril formation of the two proteins is found to be a two-stage process.

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.

Are there temperature-dependent structural transitions in the "intrinsically unstructured" protein prothymosin alpha?

Prothymosin alpha, a typical member of the class of the so-called "intrinsically unstructured" proteins, adopts a random-chain conformation under physiological environmental conditions. An apparent formation of ordered secondary structure and a moderate compaction are observed upon the change from neutral to acid pH at room temperature. We have addressed the question of whether there are temperature-dependent changes of the conformational state of prothymosin alpha at low pH using circular dichroism spectroscopy and static and dynamic light scattering.

Assembly of amyloid protofibrils via critical oligomers--a novel pathway of amyloid formation.

The amyloid formation of phosphoglycerate kinase (PGK) was investigated by static and dynamic light-scattering. The time-course of the scattering intensity and the hydrodynamic radius scale with initial monomer concentration in a linear fashion over a range of about 50 in concentration. This sets limits on theories for aggregation kinetics that can be used, and points towards irreversible, cascade type models.

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

The conformational transitions of bovine beta-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.

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.

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.

Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A.

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v).

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state.

Initial hydrophobic collapse is not necessary for folding RNase A.

Background: One of the main distinctions between different theories describing protein folding is the predicted sequence of secondary structure formation and compaction during the folding process. Whether secondary structure formation precedes compaction of the protein molecules or secondary structure formation is driven by a hydrophobic collapse cannot be decided unequivocally on the basis of existing experimental data.

Results: In this study, we investigate the refolding of chemically denatured, disulfide-intact ribonuclease A (RNase A) by monitoring compaction and secondary structure formation using stopped-flow dynamic light scattering and stopped-flow CD, respectively.

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.

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly.

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.

Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy.

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses.

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.

Reduced-denatured ribonuclease A is not in a compact state.

Dynamic light scattering and circular dichroism experiments were performed to determine the compactness and residual secondary structure of reduced and by 6 M guanidine hydrochloride denatured ribonuclease A. We find that reduction of the four disulphide bonds by dithiothreitol at 20 degrees C leads to total unfolding and that a temperature increase has no further effect on the dimension. The Stokes' radius of ribonuclease A at 20 degrees C is R(s) = (1.

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).

Cold denaturation of yeast phosphoglycerate kinase: which domain is more stable?

Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C-terminal domain should be more stable than the N-terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view.

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.

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.

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.

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.

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.

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.

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.