A chemical investigation of the organic paint binders of the Giant Buddhas of Bāmiyān was performed using an analytical approach based on mass spectrometry, combining traditional gas chromatography/mass spectrometry protocols with advanced proteomics methodologies. The research was carried out on a selection of rescued fragments. The data revealed the use of egg proteins as the paint binders of the original layers, in accordance with the traditional use of this proteinaceous medium in antiquity, spanning from the Mediterranean basin to the Far East, and already in the Bronze Age. Egg tempera was thus known to artists of the region in the first centuries AD, probably also due to the position of the Bāmiyān valley, which was connected to the Silk Road. Milk was found in the first historical overpaintings. A new proteomics approach was used, which was able to identify the source of the milk proteins present in the restoration layers, despite their age and degradation. In particular cow's and goat's milk were both found, in agreement with the documented presence of rich pastures in the Bāmiyān valley when the historical restorations were carried out. Investigating the materials of the Giant Buddhas not only enabled us to obtain isolated data on these invaluable works of art, which are now lost, but contributes to understanding the big "puzzle" of our past and the development of our culture, by implementing and supporting written sources, stylistic and anthropological studies with molecular data.
Abstract Animal glues have been used for centuries, but their popularity decreased in the 20th century with the rise of synthetic adhesives, leading to their current primary use in restoration. Despite this decline, gelatine, derived from denatured and partially hydrolysed collagen, has gained popularity in various applications. This study focuses on gelatinous glue samples derived from animal bone and hide tissues, examining their secondary structure and thermal properties to identify structure-property correlations. Infrared spectroscopy analysis has revealed differences in the secondary structures, with hide glues exhibiting more β-structures than bone glues, indicating a higher degree of aggregation. Thermogravimetric analysis and differential scanning calorimetry also have highlighted differences between hide and bone glues, showing that the latter are more hydrolysed. Furthermore, the calorimetric curves have showed different values of denaturation enthalpy thus indicating a different degree of gelatine renaturation. Additionally, the calorimetric analysis has demonstrated the physical ageing of gelatinous glue samples, a key factor in maintaining adhesive properties for long-term use under specific storage conditions. In a context prioritizing the use of waste biomass over fossil fuels, understanding the properties of gelatine in glues is crucial for enhancing their performance and promoting their adoption as sustainable alternatives to non-renewable adhesives.
Proteomic strategies are herein proved to be a complementary approach to the well established amino acid composition analysis for the characterization of the aging and deterioration phenomena occurring to proteinaceous materials in works-of-art. Amino acid analyses on several samples demonstrated that proteins in the frescoes from the Camposanto Monumentale in Pisa are deteriorated as revealed by the decrease in Met, Lys, and Tyr content and by the presence in all the samples of amino malonic acid as a result of Ser, Phe, and Cys oxidation. Proteomic analysis identified deamidation at Asn and Gln as a further major event occurred. This work paves the way to the exploitation of proteomic strategies for the investigation of the molecular effects of aging and deterioration in historical objects. Results show that proteomic searches for deamidation by liquid chromatography−tandem mass spectrometry (LC−MS/MS) could constitute a routine analysis for paintings or any artistic and historic objects where proteins are present. Peptides that can be used as molecular markers when casein is present were identified.
IF1, the natural inhibitor protein of FOF1ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [Cabezon, E., Butler, P. J., Runswick, M. J., and Walker, J. E. (2000) J. Biol. Chem. 275, 25460−25464]. IF1 is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF1 and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF1 within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF1. Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF1−CaM complex is 1:1, thereby indicating that binding to CaM promotes IF1 dimer dissociation without directly interfering with the intersubunit contacts of the IF1 dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF1−CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.
Aspartate aminotransferase from Sulfolobus solfataricus (AspATSs) is an extremely thermophilic and thermostable enzyme. In order to investigate the structural features which underlie thermophilicity and thermostability, two isoforms of AspATSs differing by a single amino acid residue were compared. The first isoform is the naturally occurring enzyme, whereas the second is a genetically engineered mutant. Thermophilicity, short‐term and long‐term thermostability of the isoenzymes were independently evaluated and the influence of a cysteine residue on the three properties was assessed.
Abstract ICOSL/ICOS are costimulatory molecules pertaining to immune checkpoints; their binding transduces signals having anti-tumor activity. Osteopontin (OPN) is here identified as a ligand for ICOSL. OPN binds a different domain from that used by ICOS, and the binding induces a conformational change in OPN, exposing domains that are relevant for its functions. Here we show that in vitro, ICOSL triggering by OPN induces cell migration, while inhibiting anchorage-independent cell growth. The mouse 4T1 breast cancer model confirms these data. In vivo, OPN-triggering of ICOSL increases angiogenesis and tumor metastatization. The findings shed new light on ICOSL function and indicate that another partner beside ICOS may be involved; they also provide a rationale for developing alternative therapeutic approaches targeting this molecular trio.
