In vitro evidence of propagation of superoxide dismutase-1 protein aggregation in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disorder that has been linked to mutations in the superoxide dismutase 1 (SOD1) gene. The accumulation of misfolded protein aggregates in spinal neurons and astrocytes is implicated as an important pathological process in DM; however, the mechanism of protein aggregate formation is largely unknown. In human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), cell-to-cell propagation of disease-relevant proteins has been demonstrated. Therefore, in this study, propagation of aggregation-forming property of mutant SOD1 protein in DM in vitro was investigated. This study demonstrated that aggregates composed of canine wild type SOD1 protein were increased by co-transfection with canine mutant SOD1 (E40K SOD1), indicating intracellular propagation of SOD1 aggregates. Further, aggregated recombinant SOD1 proteins were released from the cells, taken up by other cells, and induced further aggregate formation of normally folded SOD1 proteins. These results suggest intercellular propagation of SOD1 aggregates. The hypothesis of cell-to-cell propagation of SOD1 aggregates proposed in this study may underly the progressive nature of DM pathology.

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.

Response of native and denatured hen lysozyme to high pressure studied by (15)N/(1)H NMR spectroscopy.

High-pressure (15)N/(1)H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 M urea, over the pressure range 30--2000 bar. Most (1)H and (15)N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 +/- 0.101 p.p.m. ((1)H) and 0.51 +/- 0.36 p.p.m. ((15)N). The shifts indicate that the hydrogen bonds formed to carbonyl groups or water molecules by the backbone amides are, on average, shortened by approximately 0.02 A as a result of pressure. In native lysozyme, six residues in the beta domain or at the alpha/beta domain interface have anomalously large nonlinear (15)N and (1)H chemical-shift changes. All these residues lie close to water-containing cavities, suggesting that there are conformational changes involving these cavities, or the water molecules within them, at high pressure. The pressure-induced (1)H and (15)N shifts for lysozyme denatured in 8 M urea are much more uniform than those for native lysozyme, with average backbone amide shifts of 0.081 +/- 0.029 p.p.m. ((1)H) and 0.57 +/- 0.14 p.p.m. ((15)N). The results show that overall there are no significant variations in the local conformational properties of denatured lysozyme with pressure, although larger shifts in the vicinity of a persistent hydrophobic cluster indicate that interactions in this part of the sequence may rearrange. NMR diffusion measurements demonstrate that the effective hydrodynamic radius of denatured lysozyme, and hence the global properties of the denatured ensemble, do not change detectably at high pressure.

A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein.

Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. An extensive analysis of the denatured state at pH 8.0 was performed using a variety of techniques including (1)H nuclear magnetic resonance spectroscopy and solution X-ray scattering. This analysis indicates that the denatured state under these conditions has a compact and globular conformation with a substantial amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible region. The NMR measurements suggest that the folded region contains His53 and is located at least partly in the N-terminal lobe of the protein. The alkaline-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are pH 11.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The present findings suggest that the proteolytic processing of pepsinogen has substantially modified the ability of the protein to fold, such that its folding process cannot progress beyond the partially folded intermediate of pepsin.

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent.

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.

The methanol-induced transition and the expanded helical conformation in hen lysozyme.

Methanol-induced conformational transitions of hen egg white lysozyme were investigated with a combined use of far- and near-UV CD and NMR spectroscopies, ANS binding and small-angle X-ray scattering. Addition of methanol induced no global change in the native conformation itself, but induced a transition from the native state to the denatured state which was highly cooperative, as shown by the coincidence of transition curves monitored by the far- and near-UV CD spectroscopy, by isodichroic points in the far- and near-UV CD spectra and by the concomitant disappearance of individual 1H NMR signals of the native state. The ANS binding experiments could detect no intermediate conformer similar to the molten globule state in the process of the methanol denaturation. However, at high concentration of methanol, e.g., 60% (v/v) methanol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far-UV CD spectroscopy, and had no specific tertiary structure, monitored both by near-UV CD and NMR spectroscopy. The radius of gyration in the H state, 24.9 angstroms, was significantly larger than that in the native state (15.7 angstroms). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea-denatured state, indicating a complete lack of globularity. Thus we conclude that the H state has a considerably expanded, flexible broken rod-like conformation which is clearly distinguishable from the "molten globule" state. The stability of both N and H states depends on pH and methanol concentration. Thus a phase diagram involving N and H was constructed.

Urea-induced conformational changes in cold- and heat-denatured states of a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.

The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering.

Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering. At pH 3.0 and 25 degrees C, two methanol-induced non-native states were characterized. First, addition of methanol up to 25% (v/v) induced a compact denatured conformer (I(M)). Further addition of methanol transformed this I(M) state into the expanded and highly helical denatured state (H). The existence of the I(M) state was shown by the discrepancy in transition curves obtained from the ellipticity at 222 nm, the ellipticity at 282 nm, the tryptophan fluorescence monitored at 350 nm and the native peak intensity of the (1)H NMR spectrum. These CD, fluorescence and NMR results showed that the I(M) state has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state. The radius of gyration of the I(M) state, 17.7 angstroms, obtained from the Guinier plot of the small-angle X-ray scattering data was significantly smaller than that of the acid-denatured state (30.1 angstroms) and was closer to that of the native state (14.6 angstroms), showing that the I(M) state is compact. The Kratky plot for the I(M) state exhibited a bell-shaped profile, indicating a globular conformation. These structural features indicate that the structure of the I(M) state is quite similar to that of the anion-induced molten globule state of this protein. Furthermore the alcohol-denatured state (H) of cyt C in 60% (v/v) methanol was structurally characterized. Though the H state had a helical content much higher than the native state monitored by far-UV CD spectroscopy, the radius of gyration, 31.7 angstroms, was similar to that of the acid-denatured state, showing that this H state is an expanded denatured state. The Kratky plot for the H state did not show a clear peak, indicating a chain-like conformation. Thus we conclude that the H state has an expanded and chain-like conformation with a high helical content. Finally, we constructed a phase diagram of cyt c involving the native, I(M), acid-denatured and H states against pH and the methanol concentration. The result indicates that the I(M) state is found in the pH range from 2.5 to at least 4.5 with a pH-dependent optimum methanol concentration of 10 to 40%.