Biology subjects

Caramello, N.

Publications and source records attributed to Caramello, N..

3 recordsLinked to original sources

bioRxiv2026

Time-resolved functional rotation crystallography reveals protein dynamics and catalysis

Time-resolved crystallography offers a window into the transient, atomic-scale structural changes that underlie biological function. Here we introduce Time-Resolved Functional Rotation Crystallography (TR-FRX), a method that enables molecular-level studies of ligand binding and enzymatic catalysis using single protein crystals at room temperature. Unlike many state-of-the-art time-resolved methodologies, TR-FRX relies on standard rotation-based X-ray data collection and does not require serial sample delivery strategies. By dispensing nanoliter-scale droplets of ligand or substrate directly onto crystals mounted in conventional holders, TR-FRX captures real-time structural snapshots while maintaining experimental accessibility. As a proof-of-concept, we first monitor the binding of N-acetylglucosamine to hen egg-white lysozyme, demonstrating that TR-FRX can reveal ligand recognition in crystallo on sub-second timescales. We then resolve the bidirectional catalytic mechanism of a prototypical enzyme from the tricarboxylic-acid-cycle, revealing sequential cofactor and substrate binding, catalytic loop dynamics, and substrate stabilization across time points spanning from 137 milliseconds to minutes. Requiring only micrograms of protein and standard beamline infrastructure, TR-FRX provides an accessible methodology for capturing transient enzymatic states and enables 100-ms timescale studies at room temperature on single protein crystals.

biochemistry↗

bioRxiv2024

CryoRhodopsins: a comprehensive characterization of a new clade of microbial rhodopsins from cold environments

Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.

biophysics↗

bioRxiv2023

A redox switch allows binding of ferrous and ferric ions in the cyanobacterial iron binding protein FutA from Prochlorococcus

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance StatementOceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

biophysics↗