DNP-Enhanced MAS NMR of Bovine Serum Albumin Sediments and Solutions

[Image: see text] Protein sedimentation sans cryoprotection is a new approach to magic angle spinning (MAS) and dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy of proteins. It increases the sensitivity of the experiments by a factor of ∼4.5 in comparison to the conventional DNP sample preparation and circumvents intense background signals from the cryoprotectant. In this paper, we investigate sedimented samples and concentrated frozen solutions of natural abundance bovine serum albumin (BSA) in the absence of a glycerol-based cryoprotectant. We observe DNP signal enhancements of ε ∼ 66 at 140 GHz in a BSA pellet sedimented from an aqueous solution containing the biradical polarizing agent TOTAPOL and compare this with samples prepared using the conventional protocol (i.e., dissolution of BSA in a glycerol/water cryoprotecting mixture). The dependence of DNP parameters on the radical concentration points to the presence of an interaction between TOTAPOL and BSA, so much so that a frozen solution sans cryoprotectant still gives ε ∼ 50. We have studied the interaction of BSA with another biradical, SPIROPOL, that is more rigid than TOTAPOL and has been reported to give higher enhancements. SPIROPOL was also found to interact with BSA, and to give ε ∼ 26 close to its maximum achievable concentration. Under the same conditions, TOTAPOL gives ε ∼ 31, suggesting a lesser affinity of BSA for SPIROPOL with respect to TOTAPOL. Altogether, these results demonstrate that DNP is feasible in self-cryoprotecting samples.