Eaton, G. R. | bioRxiv Science

bioRxiv2023

Cucurbituril Enhances Distance Measurements of Spin-Labeled Proteins

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 x 106 M-1, is at least one order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase of the rotational correlation time,{tau} rot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in{tau} rot and electron spin coherence time, Tm, along with the narrowing of inter-spin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4 - 9, compared to the common spin label such as MTSL, which is not affected by CB-7. Inter-spin distances of 3-nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 [A] resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating undisturbed structure and conformation of the protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/554361v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1171bd0org.highwire.dtl.DTLVardef@231612org.highwire.dtl.DTLVardef@17e22e5org.highwire.dtl.DTLVardef@250f1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Trehalose as an alternative to glycerol as a glassing agent for in vivo DNP MRI

In dynamic nuclear polarization (DNP), the solutions of the hyperpolarizable molecule and the paramagnetic agent need to form a glass when frozen to attain significant levels of polarization in reasonable time periods. Molecules which do not form glasses by themselves are often mixed with excipients to form glasses. While glassing agents are often essential in DNP studies, they have the potential to perturb the metabolic measurements that are being studied. Glycerol, the glassing agent of choice for in vivo DNP studies, is effective at reducing ice crystal formation during freezing but is rapidly metabolized, potentially altering the redox and ATP balance of the system. As a biologically inert alternative to glycerol, we show here that 15-20 wt % trehalose yields a glass that polarizes samples more rapidly than the commonly used 60% wt formulation of glycerol and yields similar polarization levels within clinically relevant timeframes. Trehalose may be an attractive alternative to glycerol for situations where there may be concerns about glycerols glucogenic potential and possible alteration of the ATP/ADP and redox balance.

biophysics↗