Characterization of Manufactured Binding Sites in BPTI Using Laser Polarized $^{129}$Xe*

We measure the NMR chemical shift of laser polarized $^{129}$Xe in wild type, Y35G, Y23A, and F45S BPTI (Bovine Pancreatic Trypsin Inhibitor) solutions of varying concentration. Our technique uses $^{129}$Xe in unprecedented low concentrations as a biosensor. The results provide structural information concerning the aforementioned proteins [2,3]. We use a flow-through polarizer that outputs $^{129}$Xe hyperpolarized to $\sim $10{\%}. Hyperpolarized gas coupled with a high resolution NMR spectrometer, enables us to measure sub-ppm chemical shifts at very low Xe and protein concentrations. In accordance with the fast exchange regime, we observe a single resonance that is chemically shifted as a function of protein concentration. Consistent with a rigid lattice and a manufactured binding site, Y23A and F45S demonstrate strong binding relative to wild type and Y35G. Wild type is believed not to have a specific binding site. Other experiments on Y35G have demonstrated a hydrophobic cavity and extensive solution-phase motion [1]. Weak binding supports the notion that a small fraction of solution-phase Y35G is in a conformation such that the cavity is accessible to Xe. [1] S. A. Beeser, \textit{J. Mol. Biol.}, \textbf{269}, 154-164v [2] W. M. Hanson \textit{et al.}, \textit{J. Mol. Biol.} 2007, \textbf{366}, 230-243 [3] A. T. Danishefsky \textit{et al.}, \textit{Protein Sci.} 1993, \textbf{2}, 577-587