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57 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Structural and antigen-binding surface definition of an anti-CD93 monoclonal antibody for the treatment of degenerative vascular eye diseases Luisa Raucci , Cosimo Damiano Perrone , Stefano Barbera , Laurens Julius De Boer , Gian Marco Tosi , Jlenia Brunetti , Luisa Bracci , Cecilia Pozzi , Federico Galvagni , Maurizio Orlandini International Journal Of Biological Macromolecules 77 DOI: 10.1016/j.ijbiomac.2025.143118 Diamond Proposal Number(s): [36130] Open Access Show Abstract ▼ Abstract: CD93 is a receptor predominantly expressed on the surface of endothelial cells, where it plays a pivotal role in angiogenesis through its interaction with the extracellular matrix. In our previous studies, we identified the monoclonal antibody 4E1 as a potent inhibitor of angiogenesis by targeting the CD93-Multimerin-2 axis. Here, we report the development of 4E1 as a recombinant whole immunoglobulin and a single-chain variable fragment, designated sc-4E. Both formats retained the binding properties of the parental monoclonal antibody and exhibited comparable inhibitory effects on endothelial cell migration and differentiation. To elucidate the molecular basis of the 4E1-CD93 interaction, we initially employed machine learning-based modeling and docking analyses of the variable heavy and light domains of 4E1. Subsequent crystallographic analysis of sc-4E provided high-resolution structural insights, confirming and validating the predicted model. Further docking experiments and molecular dynamics simulations using the crystallographic structures of CD93 and sc-4E revealed that the interaction is primarily mediated by the CDR-H3 and CDR-L2 loops. Notably, these regions engage with the sushi-like domain of CD93, which is critical for its interaction with Multimerin-2. This comprehensive structural and functional characterization of 4E1 and sc-4E underscores their potential as anti-angiogenic agents. By effectively inhibiting endothelial cell migration and differentiation, 4E1 derivatives represent promising therapeutic candidates for the treatment of ocular vascular diseases driven by pathological angiogenesis.	Apr 2025
I22-Small angle scattering & Diffraction	Thermally and base-triggered “debond-on-demand” chain-extended polyurethane adhesives Matthew J. Hyder , Jessica Godleman , Ann M. Chippindale , James E. Hallett , Thomas Zinn , Josephine L. Harries , Wayne Hayes Macromolecules DOI: 10.1021/acs.macromol.4c02775 Diamond Proposal Number(s): [36844] Open Access Show Abstract ▼ Abstract: A series of novel chain-extended polyurethanes (CEPUs) featuring degradable sulfonyl ethyl urethane chain-extenders that permit degradation under base-triggered conditions to afford “debond-on-demand” elastomeric adhesives are reported. Exposure of the CEPUs to tetra-butylammonium fluoride (TBAF) triggered the degradation of the sulfonyl ethyl urethane chain-extenders. Lap shear adhesion tests of the CEPUs exposed to TBAF revealed reductions in shear strength of up to 65% for both aluminum and glass substrates, from 2.18 to 0.76 MPa and from 1.13 to 0.52 MPa, respectively. The selective depolymerization of these polymers makes them suitable candidates as debondable binders for inkjet inks and coatings, enabling removal of inks and adhesive residues from substrates before they enter the recycling process, to prevent surface contaminants decreasing the quality of the recycled material.	Jan 2025
I04-Macromolecular Crystallography	A low-barrier proton shared between two aspartates acts as a conformational switch that changes the substrate specificity of the β-lactamase BlaC Jing Sun , Aimee L. Boyle , Steffen Brünle , Marcellus Ubbink International Journal Of Biological Macromolecules 278 DOI: 10.1016/j.ijbiomac.2024.134665 Diamond Proposal Number(s): [25413] Open Access Show Abstract ▼ Abstract: Serine β-lactamases inactivate β-lactam antibiotics in a two-step mechanism comprising acylation and deacylation. For the deacylation step, a water molecule is activated by a conserved glutamate residue to release the adduct from the enzyme. The third-generation cephalosporin ceftazidime is a poor substrate for the class A β-lactamase BlaC from Mycobacterium tuberculosis but it can be hydrolyzed faster when the active site pocket is enlarged, as was reported for mutant BlaC P167S. The conformational change in the Ω-loop of the P167S mutant displaces the conserved glutamate (Glu166), suggesting it is not required for deacylation of the ceftazidime adduct. Here, we report the characterization of wild type BlaC and BlaC E166A at various pH values. The presence of Glu166 strongly enhances activity against nitrocefin but not ceftazidime, indicating it is indeed not required for deacylation of the adduct of the latter substrate. At high pH wild type BlaC was found to exist in two states, one of which converts ceftazidime much faster, resembling the open state previously reported for the BlaC mutant P167S. The pH-dependent switch between the closed and open states is caused by the loss at high pH of a low-barrier hydrogen bond, a proton shared between Asp172 and Asp179. These results illustrate how readily shifts in substrate specificity can occur as a consequence of subtle changes in protein structure.	Oct 2024
I02-Macromolecular Crystallography	Structural dynamics of the TPR domain of the peroxisomal cargo receptor Pex5 in Trypanosoma Michal Banasik , Valeria Napolitano , Artur Blat , Karim Abdulkarim , Jacek Plewka , Cezary Czaplewski , Artur Gieldon , Maciej Kozak , Benedykt Wladyka , Grzegorz Popowicz , Grzegorz Dubin International Journal Of Biological Macromolecules 465 DOI: 10.1016/j.ijbiomac.2024.135510 Show Abstract ▼ Abstract: Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small – angle X – ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo.	Sep 2024
B21-High Throughput SAXS	Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies Lorenzo Briganti , Livia R. Manzine , Caio Cesar De Mello Capetti , Evandro Ares De Araújo , Vanessa De Oliveira Arnoldi Pellegrini , Francisco Eduardo Gontijo Guimaraes , Mario De Oliveira Neto , Igor Polikarpov International Journal Of Biological Macromolecules 87 DOI: 10.1016/j.ijbiomac.2024.133182 Show Abstract ▼ Abstract: Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-β-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a − 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.	Jun 2024
I02-Macromolecular Crystallography	Functional and structural characterisation of RimL from Bacillus cereus, a new Nα-acetyltransferase of ribosomal proteins that was wrongly assigned as an aminoglycosyltransferase H. Leonardo Silvestre , J. L. Asensio , T. L. Blundell , A. Bastida , V. M. Bolanos-Garcia International Journal Of Biological Macromolecules 263 DOI: 10.1016/j.ijbiomac.2024.130348 Diamond Proposal Number(s): [9007, 14043] Open Access Show Abstract ▼ Abstract: Enzymes of the GNAT (GCN5-relate N-acetyltransferases) superfamily are important regulators of cell growth and development. They are functionally diverse and share low amino acid sequence identity, making functional annotation difficult. In this study, we report the function and structure of a new ribosomal enzyme, Nα-acetyl transferase from Bacillus cereus (RimLBC), a protein that was previously wrongly annotated as an aminoglycosyltransferase. Firstly, extensive comparative amino acid sequence analyses suggested RimLBC belongs to a cluster of proteins mediating acetylation of the ribosomal protein L7/L12. To assess if this was the case, several well established substrates of aminoglycosyltransferases were screened. The results of these studies did not support an aminoglycoside acetylating function for RimLBC. To gain further insight into RimLBC biological role, a series of studies that included MALDI-TOF, isothermal titration calorimetry, NMR, X-ray protein crystallography, and site-directed mutagenesis confirmed RimLBC affinity for Acetyl-CoA and that the ribosomal protein L7/L12 is a substrate of RimLBC. Last, we advance a mechanistic model of RimLBC mode of recognition of its protein substrates. Taken together, our studies confirmed RimLBC as a new ribosomal Nα-acetyltransferase and provide structural and functional insights into substrate recognition by Nα-acetyltransferases and protein acetylation in bacteria.	Apr 2024
I04-Macromolecular Crystallography	Insights into energy quenching mechanisms and carotenoid uptake by Orange carotenoid protein homologs: HCP4 and CTDH Jenia Sklyar , Adjélé Wilson , Diana Kirilovsky , Noam Adir International Journal Of Biological Macromolecules 1861 DOI: 10.1016/j.ijbiomac.2024.131028 Show Abstract ▼ Abstract: Photodamage to the photosynthetic apparatus by excessive light radiation has led to the evolution of a variety of energy dissipation mechanisms. A mechanism that exists in some cyanobacterial species, enables non-photochemical quenching of excitation energy within the phycobilisome (PBS) antenna complex by the Orange Carotenoid Protein (OCP). The OCP contains an active N-terminal domain (NTD) and a regulatory C-terminal domain (CTD). Some cyanobacteria also have genes encoding for homologs to both the CTD (CTDH) and the NTD (referred to as helical carotenoid proteins, HCP). The CTDH facilitates uptake of carotenoids from the thylakoid membranes to be transferred to the HCPs. Holo-HCPs exhibit diverse functionalities such as carotenoid carriers, singlet oxygen quenchers, and in the case of HCP4, constitutive OCP-like energy quenching. Here, we present the first crystal structure of the holo-HCP4 binding canthaxanthin molecule and an improved structure of the apo-CTDH from Anabaena sp. PCC 7120. We propose here models of the binding of the HCP4 to the PBS and the associated energy quenching mechanism. Our results show that the presence of the carotenoid is essential for fluorescence quenching. We also examined interactions within OCP-like species, including HCP4 and CTDH, providing the basis for mechanisms of carotenoid transfer from CTDH to HCPs.	Mar 2024
I22-Small angle scattering & Diffraction	Mechanistic insights into polymerization-induced self-assembly using maleimide-based fluorophores Amanda K. Pearce , Sam J. Parkinson , Irem Akar , Matthew J. Derry , Paul D. Topham , Robert T. Mathers , Vasilios G. Stavros , Rachel K. O'Reilly Macromolecules DOI: 10.1021/acs.macromol.3c01451 Diamond Proposal Number(s): [28511] Open Access Show Abstract ▼ Abstract: Polymerization-induced self-assembly (PISA) is a versatile and readily accessible method to produce nanoparticles of various morphologies in situ as polymerization progresses. PISA exploits the chain extension of a solvophilic macromolecular chain-transfer agent with monomers that are miscible in the continuous phase but form a solvophobic, immiscible polymer, driving self-assembly. However, the ability to monitor in situ the onset of self-assembly and the evolution of morphology during the PISA process remains a significant challenge, which critically limits our understanding of the mechanisms of particle formation. In this work, we demonstrate that a maleimide-based small-molecule fluorophore can act as a powerful probe to study PISA over time using fluorescence and fluorescence lifetime as outputs. We show that the aminochloromaleimide (ACM) fluorophore can be readily incorporated within a PISA system to produce fluorescent nanostructures without affecting their final morphology in comparison to their nonfluorescent analogues. The ACM probe exhibits diagnostic increases in fluorescence lifetime first with the onset of self-assembly and then with the evolution of particle morphology in the order of spheres > vesicles > worms. Excitingly, monitoring the change in fluorescence lifetime in situ during PISA yielded insights into the mechanism of particle formation when targeting higher-order morphologies. Finally, we demonstrate that these maleimide-functionalized nanostructures can be used as cell imaging agents using fluorescence lifetime imaging microscopy (FLIM), whereby each morphology produces distinct lifetime decay patterns within a cell environment. Overall, we envision this becoming a powerful tool for the analysis of nanoparticle states within complex environments, inspiring further investigations of the study of PISA using this simple and accessible method.	Nov 2023
I04-Macromolecular Crystallography	Enhanced activity against a third-generation cephalosporin by destabilization of the active site of a class A beta-lactamase Jing Sun , Aleksandra Chikunova , Aimee L. Boyle , Patrick Voskamp , Monika Timmer , Marcellus Ubbink International Journal Of Biological Macromolecules 250 DOI: 10.1016/j.ijbiomac.2023.126160 Diamond Proposal Number(s): [25413] Open Access Show Abstract ▼ Abstract: The β-lactamase BlaC conveys resistance to a broad spectrum of β-lactam antibiotics to its host Mycobacterium tuberculosis but poorly hydrolyzes third-generation cephalosporins, such as ceftazidime. Variants of other β-lactamases have been reported to gain activity against ceftazidime at the cost of the native activity. To understand this trade-off, laboratory evolution was performed, screening for enhanced ceftazidime activity. The variant BlaC Pro167Ser shows faster breakdown of ceftazidime, poor hydrolysis of ampicillin and only moderately reduced activity against nitrocefin. NMR spectroscopy, crystallography and kinetic assays demonstrate that the resting state of BlaC P167S exists in an open and a closed state. The open state is more active in the hydrolysis of ceftazidime. In this state the catalytic residue Glu166, generally believed to be involved in the activation of the water molecule required for deacylation, is rotated away from the active site, suggesting it plays no role in the hydrolysis of ceftazidime. In the closed state, deacylation of the BlaC-ceftazidime adduct is slow, while hydrolysis of nitrocefin, which requires the presence of Glu166 in the active site, is barely affected, providing a structural explanation for the trade-off in activities.	Oct 2023
B21-High Throughput SAXS	Unveiling protein-protein interaction potential through Monte Carlo simulation combined with small-angle X-ray scattering Fernando T. Tanouye , Jozismar R. Alves , Francesco Spinozzi , Rosangela Itri International Journal Of Biological Macromolecules 248 DOI: 10.1016/j.ijbiomac.2023.125869 Diamond Proposal Number(s): [17570] Show Abstract ▼ Abstract: Protein interactions are investigated under different conditions of lysozyme concentration, temperature and ionic strength by means of in-solution small angle X-Ray scattering (SAXS) experiments and Monte Carlo (MC) simulations. Initially, experimental data were analysed through a Hard-Sphere Double Yukawa (HSDY) model combined with Random Phase Approximation (RPA), a closure relationship commonly used in the literature for monodisperse systems. We realized by means of MC that the HSDY/RPA modelling fails to describe the protein-protein pair potential for moderated and dense systems at low ionic strength, mainly due to inherent distortions of the RPA approximation. Our SAXS/MC results thus show that lysozyme concentrations between 2 (diluted) and 20 mg/mL (not crowded) present similar protein-protein pair potential preserving the values of surface net charge around 7 e, protein diameter of 28 Å, decay range of attractive well potential of 3 Å and a depth of the well potential varying from 1 to 5 depending on temperature and salt addition. Noteworthy, we here propose a novel method to analyse the SAXS data from interacting proteins through MC simulations, which overcomes the deficiencies presented by the use of a closure relationship. Furthermore, this new methodology of combining SAXS with MC simulations gives a step forward to investigate more complex systems as those composed of a mixture of proteins of distinct species presenting different molecular weights (and hence sizes) and surface net charges at low, moderate and very dense systems.	Sep 2023

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