松浦 滉明

Modern insulin still depends on phenol and zinc to keep the hormone stable in vials and pumps, yet both additives slow absorption and raise safety concerns. We therefore asked a simple, clinically driven question: Can we stabilize the fast‐acting T‐state of insulin without phenol/zinc by exploiting pH‐dependent water and anion binding? Using high‐resolution synchrotron crystallography (1.4–1.76 Å), we solved novel designer and acid‐stable cubic insulin structures from pH 2 to 6 in citrate–sulfate buffers and mapped solvent/anion contacts onto computational analyses. Across the acidic range, we uncovered a conserved ‘water–anion clamp’ centered on the Phe ¹ ᴮ–Asn ³ ᴮ pocket that locks insulin in its bioactive T‐conformation while neutralizing the protein's positive charge. This clamp: (i) removes the need for phenolic ligands, and (ii) keeps monomers soluble at high concentration. The structural blueprint we provide can guide formulation of phenol‐ and zinc‐free, ultra‐rapid insulin for subcutaneous pumps and high‐strength cartridges, addressing unmet needs in intensive diabetes management. By clarifying how simple buffer anions and structured water can replace traditional preservatives, our work may link atomic‐level detail to a practical therapeutic goal: faster, safer insulin delivery.

Time-resolved X-ray crystallography is undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods for uniformly initiating time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions or signalling. A widely applicable approach is the use of photocaged substrates, where the photocage is soaked into the crystal in advance and then activated using a laser pulse to provide uniform initiation of the reaction throughout the crystal. This work characterizes photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-β and dye-decolourizing peroxidase B using a fixed target sample delivery system. Laser parameters for photoactivation are systematically explored, and time-resolved structures over timescales ranging from 100 µs to 1.4 s using synchrotron and XFEL beamlines are described. The effective use of this photocage for time-resolved crystallography is demonstrated and appropriate illumination conditions for such experiments are determined.

Abstract The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

Abstract Over the last decade, Macromolecular crystallography (MX) has advanced significantly by developing brilliant micro-focus beamlines, highly sensitive and rapid-readout detectors, and sophisticated data collection systems. Automated data collection has made it possible to efficiently collect large amounts of diffraction data from microcrystals, which would be impossible to achieve manually. This has enabled high-resolution structural analysis of ultra-small crystals, efficient screening of compounds, and new MXs for structural dynamics analysis in crystal fields.

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.