Biology subjects

Hough, M. A.

Publications and source records attributed to Hough, M. A..

5 recordsLinked to original sources

bioRxiv2025

Does crossing the pond affect crystal quality?

Room-temperature X-ray diffraction experiments can facilitate investigation of protein dynamics, efficient probing of fragment binding, and time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of room-temperature X-ray diffraction data in situ directly from crystallization plates without any manipulation of protein crystals, preserving the integrity of fragile samples. While many X-ray light sources are now equipped to grow crystals on site for room-temperature experiments, to date there has been no comprehensive analysis of the effect of shipping crystals within plates at ambient temperature for in situ room-temperature data collection. In situ methods place more stringent demands on shipping, as crystallization drops must arrive both intact and properly positioned within the plate to remain in the field of view of crystal imaging systems. The latter requirement imposes more stringent limits on shipping procedures as lateral movement of <1mm may leave a droplet out of the crystal imager field of view yet is independent of the requirement for the diffraction quality of crystals to be maintained. In contrast, the equivalent methodology for cryo-cooled crystals is well established. Here we investigate the impact of transatlantic shipping of crystals generated from commercially available proteins (lysozyme, thaumatin, and thermolysin) within the same plates in which they were grown. MiTeGen In Situ-1 plates containing the protein crystals were set up at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI, Buffalo, NY, United States) and shipped to DLS (Didcot, United Kingdom). Identical plates were prepared on site at DLS in the Crystallisation Facility at Harwell. We also performed this experiment with human lysine acetyltransferase 2B (KAT2B) to validate our proposed pipeline with a typical protein crystal sample. For shipping, we utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Thermal Shipping Box (Blue Box), which can maintain temperature for a few days, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping by standard commercial courier might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study (both those shipped from UB-HWI and set up on-site at DLS), demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site. SynopsisThe effect of international shipment of protein crystals on in situ room-temperature X-ray diffraction quality.

biophysics↗

bioRxiv2024

Double crossed? Structural and computational studies of an unusual crosslinked heme in Methylococcus capsulatus cytochrome P460

Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type heme where the porphyrin is cross-linked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 heme oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460 containing hydroxylamine oxidoreductase utilises a tyrosine cross link to heme and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the Nz atom of lysine and the heme porphyrin with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the heme. Here we describe the evidence for a novel double cross link between lysine and heme in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.

biochemistry↗

bioRxiv2024

A novel glutathione peroxidase-based biosensor disentangles differential subcellular accumulation of H2O2 and lipid hydroperoxides

Hydrogen peroxide (H2O2) and lipid hydroperoxides (LOOH) are initiators and transducers of inter- and intra-cellular signaling in response to diverse environmental, pathological and developmental cues. The accumulation of both H2O2 and LOOH is often temporally and spatially coincident in tissues, but it is unknown if this coincidence extends to subcellular compartments. If distinct accumulation of different peroxides occurs at this smaller spatial scale, then it would be an important factor in signaling specificity. Fusion of the redox-sensitive (ro)GFP2 to the Saccharomyces cerevisiae (yeast) OXIDANT RECEPTOR PEROXIDASE1 (ORP1), also known as GLUTATHIONE PEROXIDASE3 (GPX3), created a now widely used biosensor that is assumed to detect H2O2 in vivo. This is despite monomeric GPX enzymes, such as ORP1/GPX3, possessing wide peroxide substrate specificities. Consequently, we confirmed in vitro that roGFP2-ORP1 is not only oxidized by H2O2, but also by phospholipid fatty acid peroxides generated in lecithin-derived liposomes by lipoxygenase-catalyzed peroxidation. This led us to doubt that roGFP2-ORP1 in vivo is specific for H2O2. To address this issue of peroxide specificity, we constructed a modified biosensor called roGFP2-synORP1. This version has greatly diminished reactivity towards phospholipid fatty acid peroxides but retains high sensitivity for H2O2. These two roGFP2-based biosensors, targeted to chloroplasts, cytosol and the nucleus, were quantitatively imaged in parallel in Nicotiana benthamiana abaxial epidermal cells experiencing high light- and herbicide-induced photo-oxidative stress. From differential patterns of oxidation of these probes, we inferred that the chloroplasts accumulated both peroxide types. In contrast, LOOH and H2O2 accumulated exclusively in the cytosol and nucleus respectively. Therefore, this suggests that the signalling networks initiated by different peroxides will have a distinct spatial component.

molecular biology↗

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↗

bioRxiv2022

Three-dimensional structure of the single domain cupredoxin AcoP

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 [A] resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

biophysics↗