Biology subjects

Crump, M. P.

Publications and source records attributed to Crump, M. P..

3 recordsLinked to original sources

bioRxiv2026

Porphyrin driven redox tuning in structurally defined de novo heme proteins

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

biochemistry↗

bioRxiv2020

Precision design of single and multi-heme de novo proteins

The de novo design of simplified porphyrin-binding helical bundles is a versatile approach for the construction of valuable biomolecular tools to both understand and enhance protein functions such as electron transfer, oxygen binding and catalysis. However, the methods utilised to design such proteins by packing hydrophobic side chains into a buried binding pocket for ligands such as heme have typically created highly flexible, molten globule-like structures, which are not amenable to structural determination, hindering precise engineering of subsequent designs. Here we report the crystal structure of a de novo two-heme binding "maquette" protein, 4D2, derived from the previously designed D2 peptide, offering new opportunities for computational design and re-engineering. The 4D2 structure was used as a basis to create a range of heme binding proteins which retain the architecture and stability of the initial crystal structure. A well-structured single-heme binding variant was constructed by computational sequence redesign of the hydrophobic protein core, assessed by NMR, and utilised for experimental validation of computational redox prediction and design. The structure was also extended into a four-heme binding helical bundle resembling a molecular wire. Despite a molecular weight of only 24kDa, imaging by CryoEM illustrated a remarkable level of detail in this structure, indicating the positioning of both the secondary structure and the heme cofactors. The design and determination of atomic-level resolution in such de novo proteins is an invaluable resource for the continued development of novel and functional protein tools.

synthetic biology↗

bioRxiv2017

The C-Terminal Domain Of ParB Is Critical For Dynamic DNA Binding And Bridging Interactions Which Condense The Bacterial Centromere

The ParB protein forms DNA bridging interactions around parS to form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring at parS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

biochemistry↗