BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy.

Circular dichroism (CD) spectroscopy is widely used to characterize the secondary structure composition of proteins. To derive accurate and detailed structural information from the CD spectra, we have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which can handle the spectral diversity of ß-structured proteins. The BeStSel webserver provides this method with useful accessories to the community with the main goal to analyze single or multiple protein CD spectra. Uniquely, BeStSel provides information on eight secondary structure components including parallel ß-structure and antiparallel ß-sheets with three different groups of twist. It overperforms any available method in accuracy and information content, moreover, it is capable of predicting the protein fold down to the topology/homology level of the CATH classification. A new module of the webserver helps to distinguish intrinsically disordered proteins by their CD spectrum. Secondary structure calculation for uploaded PDB files will help the experimental verification of protein MD and in silico modelling using CD spectroscopy. The server also calculates extinction coefficients from the primary sequence for CD users to determine the accurate protein concentrations which is a prerequisite for reliable secondary structure determination. The BeStSel server can be freely accessed at https://bestsel.elte.hu.

Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins.

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.

Phosphatidylinositol-3,4,5-trisphosphate interacts with alpha-synuclein and initiates its aggregation and formation of Parkinson's disease-related fibril polymorphism.

Lipid interaction with α-synuclein (αSyn) has been long implicated in the pathogenesis of Parkinson's disease (PD). However, it has not been fully determined which lipids are involved in the initiation of αSyn aggregation in PD. Here exploiting genetic understanding associating the loss-of-function mutation in Synaptojanin 1 (SYNJ1), a phosphoinositide phosphatase, with familial PD and analysis of postmortem PD brains, we identified a novel lipid molecule involved in the toxic conversion of αSyn and its relation to PD. We first established a SYNJ1 knockout cell model and found SYNJ1 depletion increases the accumulation of pathological αSyn. Lipidomic analysis revealed SYNJ1 depletion elevates the level of its substrate phosphatidylinositol-3,4,5-trisphosphate (PIP3). We then employed Caenorhabditis elegans model to examine the effect of SYNJ1 defect on the neurotoxicity of αSyn. Mutations in SYNJ1 accelerated the accumulation of αSyn aggregation and induced locomotory defects in the nematodes. These results indicate that functional loss of SYNJ1 promotes the pathological aggregation of αSyn via the dysregulation of its substrate PIP3, leading to the aggravation of αSyn-mediated neurodegeneration. Treatment of cultured cell line and primary neurons with PIP3 itself or with PIP3 phosphatase inhibitor resulted in intracellular formation of αSyn inclusions. Indeed, in vitro protein-lipid overlay assay validated that phosphoinositides, especially PIP3, strongly interact with αSyn. Furthermore, the aggregation assay revealed that PIP3 not only accelerates the fibrillation of αSyn, but also induces the formation of fibrils sharing conformational and biochemical characteristics similar to the fibrils amplified from the brains of PD patients. Notably, the immunohistochemical and lipidomic analyses on postmortem brain of patients with sporadic PD showed increased PIP3 level and its colocalization with αSyn. Taken together, PIP3 dysregulation promotes the pathological aggregation of αSyn and increases the risk of developing PD, and PIP3 represents a potent target for intervention in PD.

Strong acids induce amyloid fibril formation of ß2-microglobulin via an anion-binding mechanism.

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of ß2-microglobulin (ß2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of ß2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism.

Polyphosphates induce amyloid fibril formation of α-synuclein in concentration-dependent distinct manners.

Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged "KTK" segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-µM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.

Parkinson's disease is a type of amyloidosis featuring accumulation of amyloid fibrils of α-synuclein.

Many neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates in the brain. In Parkinson's disease (PD), α-synuclein (α-syn) forms such aggregates called Lewy bodies (LBs). Recently, it has been reported that aggregates of α-syn with a cross-ß structure are capable of propagating within the brain in a prionlike manner. However, the presence of cross-ß sheet-rich aggregates in LBs has not been experimentally demonstrated so far. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and found that some of them gave a diffraction pattern typical of a cross-ß structure. This result confirms that LBs in the brain of PD patients contain amyloid fibrils with a cross-ß structure and supports the validity of in vitro propagation experiments using artificially formed amyloid fibrils of α-syn. Notably, our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation of amyloid fibrils of α-syn.

Possible mechanisms of polyphosphate-induced amyloid fibril formation of ß2-microglobulin.

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using ß2-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of ß2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged ß2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of ß2m at several micromoles per liter, a similar concentration range to that in vivo. Since ß2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with ß2m electrostatically. PolyPs appear to dehydrate water molecules around ß2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of ß2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis.

Supersaturation-Dependent Formation of Amyloid Fibrils.

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute's concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

Multistep Changes in Amyloid Structure Induced by Cross-Seeding on a Rugged Energy Landscape.

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

Inorganic polyphosphate potentiates lipopolysaccharide-induced macrophage inflammatory response.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1ß, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.

Disaggregation Behavior of Amyloid ß Fibrils by Anthocyanins Studied by Total-Internal-Reflection-Fluorescence Microscopy Coupled with a Wireless Quartz-Crystal Microbalance Biosensor.

Amyloid fibrils are formed from various proteins, some of which cause the corresponding neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. It has been reported that many compounds inhibit the formation of amyloid fibrils. Anthocyanins are flavonoid pigments present in fruits and vegetables, which are known to suppress symptoms related with Alzheimer's disease. However, the influence of anthocyanins on the amyloid fibril remains unclear. Here, we succeeded in the direct monitoring of the disaggregation reaction of single amyloid ß (Aß) fibrils by anthocyanins using total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM). It is found that the disassembly activity to the Aß fibrils depends on the number of hydroxyl groups in six-membered ring B of anthocyanin, and only delphinidin-3-galactoside, possessing three hydroxyl groups there, shows high disassembly activity. Our results show the importance of the number of hydroxyl groups and demonstrate the usefulness of TIRFM-QCM as a powerful tool in studying interactions between amyloid fibrils and compounds.

Current Understanding of the Structure, Stability and Dynamic Properties of Amyloid Fibrils.

Amyloid fibrils are supramolecular protein assemblies represented by a cross-ß structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of ß-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several ß-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the ß-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.

Polyphosphates diminish solubility of a globular protein and thereby promote amyloid aggregation.

Structural changes of globular proteins and their resultant amyloid aggregation have been associated with various human diseases, such as lysozyme amyloidosis and light-chain amyloidosis. Because many globular proteins can convert into amyloid fibrils in vitro, the mechanisms of amyloid fibril formation have been studied in various experimental systems, but several questions remain unresolved. Here, using several approaches, such as turbidimetry, fluorescence and CD spectroscopy, EM, and isothermal titration calorimetry, we examined the binding of polyphosphates to hen egg-white lysozyme under acidic conditions and observed polyphosphate-induced structural changes of the protein promoting its aggregation. Our data indicate that negatively charged polyphosphates bind to protein molecules with a net positive charge. The polyphosphate-bound, structurally destabilized protein molecules then start assembling into insoluble amorphous aggregates once they pass the solubility limit. We further show that the polyphosphates decrease the solubility limit of the protein and near this limit, the protein molecules are in a labile state and highly prone to converting into amyloid fibrils. Our results explain how polyphosphates affect amorphous aggregation of proteins, how amyloid formation is induced in the presence of polyphosphates, and how polyphosphate chain length is an important factor in amyloid formation.

Heating during agitation of ß2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. ß2-Microglobulin (ß2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of ß2m in patients are observed at neutral pH, formation of ß2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that ß2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the ß2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein.

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for type-2 diabetes mellitus) was studied by electronic circular dichroism (ECD) and NMR spectroscopy. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return (e.g.; T=37 °C, pH 4.1, cE5 =250 µm, cNaCl =50 mm, t>4-6 h) that will inevitably gravitate into the amyloid state. The strong B-type far ultraviolet (FUV)-ECD spectra and an unexpectedly strong near ultraviolet (NUV)-ECD signal (Θ≈275-285   nm ) indicate that the amyloid phase of E5 is built from monomers of quasi-elongated backbone structure (φ≈-145°, ψ≈+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediates and the buildup of "toxic" early-stage oligomers leading to self-association were identified and monitored as a function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix, exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the ß-structured amyloid nucleus, thus the process follows a nucleated growth mechanism.

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein.

Invited for the cover of this issue is the group of András Perczel at Eötvös Loránd University, Budapest, Hungary and colleagues from Osaka University, Japan. The image depicts the amyloid buildup of an Exenatide derivate miniprotein (E5) monitored on a simplified hyperspace. Read the full text of the article at 10.1002/chem.201903826.

Amyloid Formation of α-Synuclein Based on the Solubility- and Supersaturation-Dependent Mechanism.

Amyloid fibrils are formed by denatured proteins when the supersaturation of denatured proteins is broken by agitation, such as ultrasonication, or by seeding, although the detailed mechanism of how solubility and supersaturation regulate amyloid formation remains unclear. To further understand the mechanism of amyloid formation, we examined α-synuclein (α-syn) amyloid formation at varying concentrations of SDS, LPA, heparin, or NaCl at pH 7.5. Amyloid fibrils were formed below or around the critical micelle concentrations (CMCs) of SDS (2.75 mM) and LPA (0.24 mM), although no fibrils were formed above the CMCs. On the other hand, amyloid fibrils were formed with 0.01-2.5 mg/mL of heparin and 0.5-1.0 M NaCl, and amyloid formation was gradually suppressed at higher concentrations of heparin and NaCl. To reproduce these concentration-dependent effects of additives, we constructed two models: (i) the ligand-binding-dependent solubility-modulation model and (ii) the cosolute-dependent direct solubility-modulation model, both of which were used by Tanford and colleagues to analyze the additive-dependent conformational transitions of proteins. The solubility of α-syn was assumed to vary depending on the concentration of additives either by the decreased solubility of the additive-α-syn complex (model i) or by the direct regulation of α-syn solubility (model ii). Both models well reproduced additive-dependent bell-shaped profiles of acceleration and inhibition observed for SDS and LPA. As for heparin and NaCl, participation of amorphous aggregates at high concentrations of additives was suggested. The models confirmed that solubility and supersaturation play major roles in driving amyloid formation in vitro, furthering our understanding of the pathogenesis of amyloidosis in vivo.

Excited-State Dynamics Affected by Switching of a Hydrogen-Bond Network in Hydrated Aminopyrazine Clusters.

The cluster structures of hydrated aminopyrazines, APz-(H2O)n=2-4, in supersonic jets have been investigated measuring the size-selected electronic and vibrational spectra and determined with the aid of quantum chemical calculations. The APz-(H2O)2 structure is assigned as a cyclic N1 type where a homodromic hydrogen-bond chain starts from the amino group and ends at the 1-position nitrogen atom of the pyrazine moiety, corresponding to 2-aminopyridine-(H2O)2. On the other hand, APz-(H2O)n=3,4 has a linear hydrogen-bond network ending at the 4-position one (N4), which resembles 3-aminopyridine-(H2O)n=3,4. The hydrogen-bond network switching from the N1 type to the N4 one provides the accompanying red shifts of the S1-S0 electronic transition that are entirely consistent with those of the corresponding 2-aminopyridine and 3-aminopyridine clusters and also shows the drastically strengthened fluorescence intensity of origin bands in the electronic spectrum. The significant change in the excited-state dynamics is explored by the fluorescence lifetime measurement and the time-dependent density functional theory (TD-DFT) calculation. It is suggested that the drastic elongation of fluorescence lifetimes is due to the change in the electronic structure of the first excited state from nπ* to ππ*, resulting in the decreasing spin-orbit coupling to T1 (ππ*).

BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra.

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 ß-sheet content is challenging because of the large spectral and structural diversity of ß-sheets. Recently, we showed that the orientation and twisting of ß-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-ß structure and antiparallel ß-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.