Biology subjects

Whittaker, J. J.

Publications and source records attributed to Whittaker, J. J..

3 recordsLinked to original sources

bioRxiv2024

Structural and biochemical analysis of a B12 super-binder

The gut microbiota is pivotal to human health, playing an important role in nutrient absorption and immune system. A prominent bacterial family in the human gut is Bacteroidetes. Cobalamin (Vitamin B12) is an essential micronutrient for these gut bacteria. Here, we focused on the BtuG family proteins in B. thetaiotaomicron, a model organism for this family, particularly the three homologs BtuG1, BtuG2, and BtuG3, which serve as cobalamin scavengers. Our study aimed to understand the structural and biochemical attributes of these proteins to describe their function in cobalamin acquisition. We solved the crystal structures of all three BtuG homologs bound to different cobalamin forms and the precursor cobinamide, and measured the binding kinetics by the grained coupled interferometry (GCI) technology. Our results reveal high binding affinities, in the low picomolar range underlining their critical role in cobalamin scavenging. Leveraging the high-resolution crystal structures, we successfully designed mutants of these BtuG homologs, resulting in both increased and decreased binding affinities for cobalamin and its precursor. This study furthers our understanding of bacterial adaptation mechanisms in the gut environment, and reveals a family of proteins with unique characteristics toward B12 scavenging, with implications for medical and biotechnological research.

biochemistry↗

bioRxiv2024

Unravelled proteins form blobs during translocation across nanopores

The electroosmotic-driven transport of unravelled proteins across nanopores is an important biological process that is now under investigation for the rapid analysis and sequencing of proteins. For this approach to work, however, it is crucial that the polymer is threaded in single file. Here we found that, contrary to the electrophoretic transport of charged polymers such as DNA, during polypeptide translocation blob-like structures typically form inside nanopores. Comparisons between different nanopore sizes, shapes and surface chemistries showed that under electroosmotic-dominated regimes single-file transport of polypeptides can be achieved using nanopores that simultaneously have an entry and an internal diameter that is smaller than the persistence length of the polymer, have a uniform non-sticky (i.e. non-aromatic) nanopore inner surface, and using moderate translocation velocities.

biophysics↗

bioRxiv2023

Legionella pneumophila macrophage infectivity potentiator protein appendage domains modulate protein dynamics and inhibitor binding

Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila, the causative agent of Legionnaires disease and protozoans such as Trypanosoma cruzi. All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl-cis/trans- isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella pneumophila protein (LpMIP) have additional appendage domains of mostly unknown function. In full- length, homodimeric LpMIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of LpMIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of LpMIP and TcMIP at 1.49 and 1.34 [A], respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms.

biophysics↗