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 β-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 β-structure and antiparallel β-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.

Biopolymere wie Proteine und Nukleinsäuren sind aus enantiomerenreinen Monomeren aufgebaut. [1] Der Ursprung des biomolekularen Symmetriebruchs -ein entscheidender Schritt für den Beginn allen Lebens auf der Erde -ist bisher nicht bekannt. Neben verschiedenen zufälligen [2] und deterministischen [3] Hypothesen schlägt ein weit verbreitetes photochemisches Modell vor, dass chirale Photonen in Form von circular polarisiertem (CP-) Licht einen Enantiomerenüberschuss in chirale organische Moleküle induzieren kçnnen. Zu diesem Modell zählt auch die enantioselektive Photolyse. [4][5][6][7] Es wird angenommen, dass asymmetrische photochemische Reaktionen interstellarer organischer Verbindungen bereits vor deren Transport auf die frühe Erde stattfanden. [8] Demzufolge interagiert interstellare CP-Strahlung, ähnlich der CP-Strahlung wie sie in der sternenbildenden Region des Orions nachgewiesen werden konnte, [5,9] asymmetrisch mit chiralen organischen Verbindungen, welche im interstellaren Eis [10] und in kohlenstoffhaltigen Meteoriten identifiziert wurden. [11] In chiralen Molekülen absorbieren beide Enantiomere die photolyseauslçsenden CP-Photonen, doch hat eines der beiden Enantiomere einen geringfügig kleineren Absorptionskoeffizienten. Dieses Enantiomer wird weniger schnell photochemisch zersetzt als sein Antipode und daher angereichert. Der dabei induzier-bare Enantiomerenüberschuss (ee) ist von der Umsatzvariablen x und dem Anisotropiefaktor g, definiert als De/e, dem Verhältnis aus differentiellem Absorptionskoeffizienten De und dem Absorptionskoeffizienten e, abhängig. Intensität und Vorzeichen von g werden allerdings durch die Wellenlänge des CP-Lichtes determiniert. Wir zeigen erstmals Anisotropiespektren von Aminosäuren als Funktion der Wellenlänge g(l), welche durch die Präparation von isotropen und amorphen Filmen im Spektralbereich zwischen 130 und 350 nm am Synchrotronzentrum ASTRID der Universität Aarhus (Dänemark) aufgenommen wurden. Die Anisotropiespektren dieser Aminosäuren in fester Phase weisen verschiedene Nulldurchgänge, Extrema und g-Werte bis zu 0.024 auf. Die Anisotropiespektren erlauben 1) die Vorhersage des Vorzeichens induzierter ee-Werte, 2) die Bestimmung von Kinetik und ee-Werten enantioselektiver Photolysereaktionen und 3) die Wahl der Wellenlänge des CP-Lichtes, die einen Enantiomerenüberschuss zu induzieren vermag.Die enantioselektive Photolyse einer racemischen Mischung durch CP-Licht entspricht einer asymmetrischen Transformation, welche durch zwei konkurrierende Reaktionen pseudo-erster Ordnung mit den Geschwindigkeitskonstanten k R und k S für das R-und S-Enantiomer beschrieben werden kann. [4] Die Geschwindigkeitskonstanten verhalten sich proportional zu ihren molaren Absorptionskoeffizienten (e R und e S ). Die Effizienz einer enantioselektiven Photolyse ist abhängig von der Differenz aus k R und k S oder, wie in diesem Falle bereits von Kuhn aufgezeigt, [12] abhängig vom Anisotropiefaktor g [Gl.(1)]. [4,6] Erst kürzlich bewiesen Nakamura et al., dass Gleichung (1) ebenso für Rea...

Intrinsically disordered proteins lack a stable tertiary structure and form dynamic conformational ensembles due to their characteristic physicochemical properties and amino acid composition. They are abundant in nature and responsible for a large variety of cellular functions. While numerous bioinformatics tools have been developed for in silico disorder prediction in the last decades, there is a need for experimental methods to verify the disordered state. CD spectroscopy is widely used for protein secondary structure analysis. It is usable in a wide concentration range under various buffer conditions. Even without providing high-resolution information, it is especially useful when NMR, X-ray, or other techniques are problematic or one simply needs a fast technique to verify the structure of proteins. Here, we propose an automatized binary disorder–order classification method by analyzing far-UV CD spectroscopy data. The method needs CD data at only three wavelength points, making high-throughput data collection possible. The mathematical analysis applies the k-nearest neighbor algorithm with cosine distance function, which is independent of the spectral amplitude and thus free of concentration determination errors. Moreover, the method can be used even for strong absorbing samples, such as the case of crowded environmental conditions, if the spectrum can be recorded down to the wavelength of 212 nm. We believe the classification method will be useful in identifying disorder and will also facilitate the growth of experimental data in IDP databases. The method is implemented on a webserver and freely available for academic users.

Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient’s sera and with mouse monoclonal antibodies. Disruption of the C28–C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13–C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.

Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-l-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism’s health, we use chiral-selective in vivo assays to detect protein-bound non-l-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. We find that altering the in vivo chiral balance creates a ‘heterochirality syndrome’ with impaired caspase activity, increased tumour formation, and premature death. Our work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.

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Amyloids are supramolecular protein assemblies based on fibrillar arrangements of βsheets that were first found as linked to neurodegenerative and systemic human diseases. However, there is now overwhelming evidence on alternative roles of amyloids as functional assemblies and as epigenetic determinants of beneficial traits, both in Fungi and Metazoa. Bacteria also use amyloids as functional devices, mainly as extracellular scaffolds in biofilms, but there is increasing evidence for functional roles of amyloids in the bacterial cytosol, and these have enabled to engineer minimal models of a 'generic' amyloid disease. Amyloids are thus key players in the physiology of bacteria and versatile building blocks in Synthetic Biology.

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