Biology subjects

Wilhelm, K. B.

Publications and source records attributed to Wilhelm, K. B..

3 recordsLinked to original sources

bioRxiv2022

A membrane-associated light harvesting model is enabled by functionalized assemblies of gene-doubled TMV proteins

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light harvesting complexes embedded within phospholipid membranes. Artificial light harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Most artificial light harvesting complexes are static or in the solution phase, rather than in a two-dimensional fluid environment as in natural photosynthesis. We have developed a method for attaching a protein-based light harvesting model to a supported lipid bilayer (SLB), which provides an extended fluid membrane surface stably associated with a solid substrate. The protein model consisted of the tobacco mosaic viral capsid proteins (TMV) that were gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV were shown to break the facial symmetry of the double disk to allow for differentiation between the disk faces. Single reactive lysine and cysteine residues were incorporated into opposing surfaces of each monomer of the dTMV assemblies. This allowed for the site-selective attachment of both chromophores for light absorption and a peptide for attachment to the SLB. A cysteine modification strategy using the enzyme tyrosinase was employed for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes showed significant association with SLBs and exhibited mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light harvesting system. Significance StatementHere we have constructed a model photosynthetic membrane containing proteins, chromophores, lipids, and aqueous components, all of which can be modified in their composition. This model is based on an asymmetric disk assembly consisting of engineered tandem dimers of the tobacco mosaic viral capsid protein (dTMV). We have developed methods to achieve dye conjugation and attachment of a supported lipid bilayers (SLB) site selectively on distinct protein surfaces. These dye-labeled protein complexes exhibit mobility on the SLB, resulting in a dynamic model of light harvesting membranes using entirely synthetic components. Additionally, this unique asymmetric assembly of TMV and the facile methods for protein functionalization are expected to expand the tunability of model light harvesting systems.

bioengineering↗

bioRxiv2021

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-{gamma}1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a role for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

biophysics↗

bioRxiv2021

Height, but not binding epitope, affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-MHC (pMHC) molecules can trigger T-cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCR can also be activated by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit non-physiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCR{beta} Fab antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab-DNA ligand efficiently activates TCR as a monomer when membrane-associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for effective TCR triggering and cellular activation by Fab-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex, but that length of the DNA tether is important. Increasing the intermembrane distance spanned by Fab-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for construction of synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand. STATEMENT OF SIGNIFICANCEWe report geometric requirements for potent T cell activation by synthetic TCR ligands that mimic biophysical properties of the native pMHC ligand, but have the additional ability to activate polyclonal T cell populations. We find that increasing the space between apposed membranes at TCR binding events decreases ligand potency, but that changing the ligands binding epitope on the TCR has essentially no effect. The observed decrease in potency with increased ligand height is attributed to the longer ligands attenuated ability to trigger TCR at binding events.

biophysics↗