Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure. Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra. These methods often fail to provide acceptable results on α/β-mixed or β-structure-rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-sheets and accurately estimates the secondary structure for a broad range of proteins. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum. By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community. The method is especially useful when X-ray or NMR techniques fail. Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides. circular dichroism | secondary structure determination | protein fold | protein aggregation | amyloid O ptically active macromolecules, such as proteins, exhibit differential absorption of circular polarized light. The far-UV circular dichroism (CD) spectroscopy of proteins and peptides (180-250 nm) is predominantly based on the excitation of electronic transitions in amide groups. The peptide backbone forms characteristic secondary structures such as α-helices, β-pleated sheets, turns, and disordered sections with specific Φ, Ψ dihedral angles and H-bond patterns affecting the CD spectrum (1). CD has been exploited for protein folding and stability assays, intermolecular interactions, and ligand binding studies, and has recently been applied in the investigations of protein disorder (2, 3). Synchrotron radiation CD (SRCD) spectroscopy is an emerging technique complementary to small-angle X-ray scattering or infrared spectroscopy, synergistic to biochemical and biophysical assays characterizing the protein folding state. SRCD extends the limits of conventional CD spectroscopy by broadening the spectral range, increasing the signal-to-noise ratio, and accelerating the data acquisition, in the presence of absorbing components (buffers, salts, etc.) (4). Additionally, SRCD has the capability of time-resolved and stopped-flow measurements as well as hig...

Circular dichroism (CD) spectroscopy is a widely used method to study the protein secondary structure. However, for decades, the general opinion was that the correct estimation of β-sheet content is challenging because of the large spectral and structural diversity of β-sheets. Recently, we showed that the orientation and twisting of β-sheets account for the observed spectral diversity, and developed a new method to estimate accurately the secondary structure (PNAS, 112, E3095). BeStSel web server provides the Beta Structure Selection method to analyze the CD spectra recorded by conventional or synchrotron radiation CD equipment. Both normalized and measured data can be uploaded to the server either as a single spectrum or series of spectra. The originality of BeStSel is that it carries out a detailed secondary structure analysis providing information on eight secondary structure components including parallel-β structure and antiparallel β-sheets with three different groups of twist. Based on these, it predicts the protein fold down to the topology/homology level of the CATH protein fold classification. The server also provides a module to analyze the structures deposited in the PDB for BeStSel secondary structure contents in relation to Dictionary of Secondary Structure of Proteins data. The BeStSel server is freely accessible at http://bestsel.elte.hu.

ABSTRACT3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17°C higher melting temperature. Its temperature optimum is 22-25°C higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen͞deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational f lexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical f lexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a ''corresponding state'' regarding conformational f lexibility. These observations confirm that conformational f luctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational f lexibility and enzyme function.

In beta(2)-microglobulin-related (Abeta2M) amyloidosis, partial unfolding of beta(2)-microglobulin (beta2-m) is believed to be prerequisite to its assembly into Abeta2M amyloid fibrils in vivo. Although low pH or 2,2,2-trifluoroethanol at a low concentration has been reported to induce partial unfolding of beta2-m and subsequent amyloid fibril formation in vitro, factors that induce them under near physiological conditions have not been determined. Using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy, we here show that at low concentrations, sodium dodecyl sulfate (SDS) converts natively folded beta2-m monomers into partially folded, alpha-helix-containing conformers. Surprisingly, this results in the extension of Abeta2M amyloid fibrils at neutral pH, which could be explained basically by a first-order kinetic model. At low concentrations, SDS also stabilized the fibrils at neutral pH. These SDS effects were concentration-dependent and maximal at approximately 0.5 mM, around the critical micelle concentration of SDS (0.67 mM). As the concentration of SDS was increased above 1 mM, the alpha-helix content of beta2-m rose to approximately 10%, while the beta-sheet content decreased to approximately 20%, a change paralleled by a complete cessation of fibril extension and the destabilization of the fibrils. Detergents of other classes had no significant effect on the extension of fibrils. These findings are consistent with the hypothesis that in vivo, specific factors (e.g., phospholipids) that affect the conformation and stability of beta2-m and amyloid fibrils will have significant effects on the kinetics of Abeta2M fibril formation.

Amyloid fibril deposition is associated with over 20 degenerative diseases, including Alzheimer's, Parkinson's, and prion diseases. Although research over the last few years has revealed the morphology and structural features of the amyloid form, knowledge about the thermodynamics of amyloid formation is limited. Here, we report for the first time a direct thermodynamic study of amyloid formation using isothermal titration calorimetry. ␤ 2 -Microglobulin, a protein responsible for dialysis-related amyloidosis, was used for extending amyloid fibrils in a seed-controlled reaction in the cell of the calorimeter. We investigated the enthalpy and heat capacity changes of the reaction, where the monomeric, acid-denatured molecules adopt an ordered, cross-␤-sheet structure in the rigid amyloid fibrils. Despite the dramatic difference in morphology, ␤ 2 -microglobulin exhibited a similar heat capacity change upon amyloid formation to that of the folding to the native globular state, whereas the enthalpy change of the reaction proved to be markedly lower. In comparison with the native state, the results outline the important structural features of the amyloid fibrils: a similar extent of surface burial even with the supramolecular architecture of amyloid fibrils, a lower level of internal packing, and the possible presence of unfavorable side chain contributions.Amyloid fibril deposition of different proteins is manifested in over 20 degenerative disorders including Alzheimer's disease, transmissible spongiform encephalopathies, and dialysisrelated amyloidosis (1-3). The increasing wealth of information accumulated in recent years has proven that the ability to form amyloid structures is a general property of the polypeptide chains. A large number of proteins and peptides have been shown to be capable of polymerization into amyloid fibrils in vitro under appropriate conditions, such as low pH, high temperature, or moderate concentrations of salts or co-solvents (1, 4 -7). Amyloid fibrils are characterized by a cross-␤-structure where ␤-strands are perpendicularly oriented to the axis of the fibrils (8). The morphology and structure of the amyloid form have been widely studied using electron microscopy, atomic force microscopy, x-ray diffraction, solid state NMR, and several spectroscopic methods (1, 2, 4 -10). Kinetic studies showed that the amyloid formation is a nucleation-dependent complex reaction (see the detailed review of Stefani and Dobson (5)).Despite the numerous studies on protein misfolding and amyloidosis, knowledge on the thermodynamics of amyloid formation is limited. Several studies have reported the structural and thermodynamic requirements of fibril formation from the side of the amyloidogenic precursor, showing the role of the stability of the native state in the amyloidogenicity or the importance of the accumulation of intermediate states (11)(12)(13). Recent studies have revealed the importance of general physicochemical characteristics of the polypeptide chain, such as the hydrophobicity, secondary ...

SignificanceSynaptic pruning is dominant in early ontogenesis when a large number of unnecessary synapses are eliminated, and it maintains synaptic plasticity in the mature healthy brain, e.g., in memory processes. Its malfunction is involved in degenerative diseases such as Alzheimer’s disease. C1q, a member of the immune complement system, plays a central role in the selective pruning of synapses by microglial phagocytosis. Understanding the molecular aspects of complement-mediated synapse elimination is of high importance for developing effective therapeutic interventions in the future. Our analysis on C1q-tagged synaptosomes revealed that C1q label-based synaptic pruning is linked to local apoptotic-like processes in synapses.

Background: Relationship between amyloid fibrils and amorphous aggregates has not yet been elucidated. Results: A competitive mechanism of amyloid fibrillation and amorphous aggregation reproduced the observed aggregation kinetics of ␤ 2 -microglobulin. Conclusion: Apparent complexities in amyloid fibrillation are explained assuming supersaturation-limited crystal-like amyloid fibrils and unlimited glass-like amorphous aggregates. Significance: Linkage of the kinetics of protein aggregation and a conformational phase diagram improves the understanding of protein aggregation.

The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.