當舎 武彦

Protein dynamics play a crucial role in various physiological functions, including enzyme catalysis. To explore conformational changes during enzyme catalysis, we conduct mix-and-inject serial crystallography, an advanced technique to capture time-resolved protein structures in real time, using the microcrystals of bacterial copper amine oxidase containing a protein-derived quinone cofactor. Within 50 ms of mixing the microcrystals (<4 μm) with a preferred substrate (2-phenylethylamine) under anaerobic conditions (reductive half-reaction), we observe domain movements associated with substrate binding and formation of a metastable reaction intermediate, a product Schiff-base of the quinone cofactor. At 100-1000 ms after mixing, conformational transition from aminoresorcinol to the semiquinone radical forms of the reduced cofactor progresses gradually, likely depending on the replacement of the product aldehyde by the next-cycle amine substrate that triggers the cofactor conformational change. Overall, this study provides structural insight into enzyme catalysis accompanying the active-site conformational changes that are hardly scrutinized by studies in solution.

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 Phe1ᴮ-Asn3ᴮ 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.

In photoactive proteins, coupling between the chromophore and protein matrix is exquisitely tuned. Proton transfer reactions can mediate this coupling, as in proton-coupled electron transfer and excited-state proton transfer. Additional mechanisms involving proton dislocations may exist but remain undiscovered. Here, we present a femtosecond crystallographic movie of the phytochrome from Deinococcus radiodurans. The structures reveal a space-conserving mechanism for rotation of the D-ring in the excited state. We observe rearrangement of a conserved hydrogen bond network within 300 fs, which precedes the isomerization reaction of the chromophore. Aided by molecular modeling and independently confirmed by femtosecond infrared spectroscopy, we attribute these changes to a protonation shift of the strictly conserved histidine-260. Although this histidine lies close to the photoexcited π-orbitals of the chromophore, it is not directly part of them. We propose that this "remote-controlled" proton transfer relays photoexcitation near-instantaneously to the protein matrix. This mechanism may be widely used to transduce cofactor signals to their hosting enzymes.

Since the birth of biochemistry, researchers have investigated the structure-function relationship of a wide variety of proteins. However, until recently, when X-ray free-electron lasers (XFELs) became available, it was not possible to visualize the motion of proteins from moment to moment with excellent temporal and spatial resolution. Here, we introduce practical methods to visualize protein motions at room temperature using serial femtosecond crystallography (SFX) using XFELs. With the development of this technology, it will be possible to visualize the entire reaction mechanism of many proteins in the future. We first outline a streamlined microcrystallization workflow for hen egg-white lysozyme, enabling rapid detector calibration and data-collection optimization. Next, we present a rotational seeding approach refined on copper-containing nitrite reductase that yields homogeneous microcrystals suitable for high-resolution SFX and readily adaptable to other challenging targets. Finally, we describe a time-resolved strategy combining microcrystals of fungal nitric-oxide reductase with photolabile caged substrates and synchronized UV triggering, capturing catalytic intermediates on the millisecond timescale. Together, these procedures enable investigators to progress from preparing samples to capturing dynamic structural snapshots. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microcrystallization of lysozyme Basic Protocol 2: Microcrystallization of copper-containing nitrite reductase Basic Protocol 3: Time-resolved serial femtosecond crystallography.