A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with 64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.
Laboratory courses are often designed
using step-by-step protocols
which encourage students to conduct experiments without thinking about
what they are doing or why they are doing it. Such course design limits
the growth of our students as scientists and can make it more difficult
for a student to transition to the expectations of a research laboratory
experience. To facilitate student growth in the process skills necessary
to transition from the teaching laboratory to the research laboratory,
an advanced biochemistry laboratory was redesigned to be team-taught
and project-based culminating in a 7 week group research project in
which the students worked collaboratively to propose, design, and
troubleshoot their own experiments. Here, we report perceived student
learning gains which provide additional evidence that inquiry- and
research-based pedagogies impact student confidence with respect to
the process skills required for self-directed research.
Structure-based drug design relies on static protein structures despite significant evidence for the need to include protein dynamics as a serious consideration. In practice, dynamic motions are neglected because they are not understood well enough to model – a situation resulting from a lack of explicit experimental examples of dynamic receptor-ligand complexes. Here, we report high-resolution details of pronounced ~1 ms timescale motions of a receptor-small molecule complex using a combination of NMR and X-ray crystallography. Large conformational dynamics in Escherichia coli dihydrofolate reductase are driven by internal switching motions of the drug-like, nanomolar-affinity inhibitor. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments and NOEs revealed the crystal structure to contain critical elements of the high energy protein-ligand conformation. The availability of accurate, structurally resolved dynamics in a protein-ligand complex should serve as a valuable benchmark for modeling dynamics in other receptor-ligand complexes and prediction of binding affinities.
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