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bioRxiv · 10.1101/2023.08.22.554361

Cucurbituril Enhances Distance Measurements of Spin-Labeled Proteins

Yang, Z.·Stein, R. A.·Pink, M.·Madzelan, P.·Ngendahimana, T.·Rajca, S.·Wilson, M. A.·Eaton, S. S.·Eaton, G. R.·Mchaourab, H. S.·Rajca, A.
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Abstract

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

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Yang, Z., Stein, R. A., Pink, M., Madzelan, P., Ngendahimana, T., Rajca, S., Wilson, M. A., Eaton, S. S., Eaton, G. R., Mchaourab, H. S., Rajca, A.. 2023-08-23. Cucurbituril Enhances Distance Measurements of Spin-Labeled Proteins. https://doi.org/10.1101/2023.08.22.554361

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