Hatton, C. E. | bioRxiv Science

bioRxiv2022

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with millisecond time-resolution. Canonical micromesh loops are mounted on an electropneumatic piston, reactions are initiated via the liquid application method (LAMA), and finally intermediate states are cryo-trapped in liquid nitrogen. We demonstrate binding of several ligands in microcrystals of three enzymes, and trapping of reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase.

biophysics↗

bioRxiv2021

Structure and mechanism of a cytosine transport protein

CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB also to be important in the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence in the organism. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4[A] resolution in complex with cytosine. We show that the protein carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 make specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.

biochemistry↗