Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in ∼12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week.National Institutes of Health (U.S.) (grant R01CA103866)National Institutes of Health (U.S.) (grant R01CA129105)National Institutes of Health (U.S.) (grant R37AI047389)United States. Department of Defense (W81XWH-15-1-0230)National Institute of General Medical Sciences (U.S.) (award Number T32GM007753

Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase and retain potency against drug-resistance mutations. Our discovery that water molecules play an essential role in the drug binding is reported. Femtosecond experiments and theory expose molecular level dynamics of rilpivirine bound to HIV-1 reverse transcriptase. The two nitrile substituents (-CN), one on each arm of the drug, have vibrational spectra consistent with their protein environments being similar in crystals and in solutions. Two-dimensional vibrational-echo spectroscopy reveals a dry environment for one nitrile while unexpectedly the other is hydrogen-bonded to a mobile water molecule, not identified in earlier X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure indeed reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond, despite the enlargement and structural variation of the binding pocket, may help retain the potency of rilpivirine against the pocket mutations