FIGURE 1.

HIV-1 Tat is monomethylated at Lys-71 by KMT7. In vitro methylation assays were done with 3 μg of synthesized Tat72 proteins that were unmodified, acetylated (K50ac), methylated (K51me), or both acetylated and methylated, with or without 1 μg of recombinant KMT7 in the presence of [³H]SAM. A, top, representative autoradiogram. This image is uncropped and from one gel; the difference in background is an imperfection of the film. Bottom, Coomassie Blue stain for Tat. Molecular weight markers are noted in kilodaltons (kDa) B, quantification of three autoradiograms with ImageJ (mean ± S.E.). C, in vitro methylation assays on unmodified Tat proteins with indicated enzymes were subjected to MS, shown are the zoomed regions containing Tat ions corresponding to aa 48–72. Boxed in red are peaks indicating modified Tat ions. D, MS/MS spectra of the ions boxed in 1C, and their corresponding peptide sequences. Ion annotations are found in supplemental Table S-1.