Nly observed. Single molecule experiments are highly selective as they focus

Nly observed. Single molecule experiments are highly selective as they focus on active enzymes only, while in bulk phase measurements an unknown fraction of inactive enzymes can distort the data. Although these figures are different from those of the bulk measurement (Tab. 1) they corroborate the finding that the bulk portion of ML 281 custom synthesis subunit c carries out ATP driven rotation despite of a rotor-stator cross-link in the mutants MM10, GH54, and FH4.DiscussionWe found that a cross-link between the top of the rotor (subunit c) and the stator ((ab)3) of F1 does not necessarily totally inhibit its ATP hydrolysis activity, but gradually reduces the rate up to fourfold (GH19), provided that the lock site on subunit c is notFigure 3. SDS-gel of the mutant GH54 under rotation assay conditions. Two samples of GH54 were oxidized (ox.) with 4 mM or 8 mM DTNB for 12 minutes, and afterwards re-reduced (re-red.) with 20 mM DTT for 12 minutes, to simulate the conditions in the rotation assay. doi:10.1371/journal.pone.0053754.gFigure 4. Rotary trajectories of reduced and oxidized F1 molecules. Trajectories of three active single molecules of GH54 driven by ATP hydrolysis both in the reduced (dashed line) and in the oxidized (dotted line) state, respectively. The mean trajectories for each of both states are shown by the solid lines. doi:10.1371/journal.pone.0053754.gUnfolding of Subunit Gamma in Rotary F-ATPasefarther than nine residues away from its C-terminal end. A crosslink at the penultimate residue of the C-terminal end (c285C, MM10) was even without any effect on the activity. In contrast, a cross-link of residues c262C (PP2, middle) or c87C (SW3, bottom) with the stator subunits practically extinguished the hydrolysis activity of F1. Three different lines of evidence support our observation. First, SDS-gels showed a cross-link yield of .85 . Second, bulk phase experiments revealed an activity of cross-linked mutants of at least 26 compared to wild type EF1 that could be restored after rereducing the samples. Third, rotation assay experiments support our conclusions on a single molecule level. Not only did we find single molecules still rotating despite oxidation, but furthermore was the rotational rate reduced by 60 , indicating that rotation was impaired by the cross-link. However, as we observed only a small number of molecules the statistics are only qualitative comparable with bulk phase experiments. What is the reason for this unusual behavior? For steric reasons alone the rotation of subunit c around the engineered disulfide bond can be excluded because that would imply the dragging around the now obliquely oriented residues 24786787 up to the C-terminal end in the before snugly fitting hydrophobic bearing. It is apparent from the crystal structure [1] (Fig. 1) that there is no space within (ab)3 for such a deformation. On the other hand, at the top subunit c is linked to a loop in subunit a that might provide some flexibility to the movement, enabling the tip of subunit c to rotate on a `leash’. However, this is very unlikely for the following reason. Czub and Grubmuller [25] have shown by molecular dynamics ?SR-3029 simulation (MD) that the respective portion of subunit c is at least four times more flexible than the opposing loop in subunit b (and therefore it is likely to be true for subunit a also because of its homology to subunit b), i.e. any torque applied would first result in a deformation of subunit c before subunit a is affected. Disulfide bond cleav.Nly observed. Single molecule experiments are highly selective as they focus on active enzymes only, while in bulk phase measurements an unknown fraction of inactive enzymes can distort the data. Although these figures are different from those of the bulk measurement (Tab. 1) they corroborate the finding that the bulk portion of subunit c carries out ATP driven rotation despite of a rotor-stator cross-link in the mutants MM10, GH54, and FH4.DiscussionWe found that a cross-link between the top of the rotor (subunit c) and the stator ((ab)3) of F1 does not necessarily totally inhibit its ATP hydrolysis activity, but gradually reduces the rate up to fourfold (GH19), provided that the lock site on subunit c is notFigure 3. SDS-gel of the mutant GH54 under rotation assay conditions. Two samples of GH54 were oxidized (ox.) with 4 mM or 8 mM DTNB for 12 minutes, and afterwards re-reduced (re-red.) with 20 mM DTT for 12 minutes, to simulate the conditions in the rotation assay. doi:10.1371/journal.pone.0053754.gFigure 4. Rotary trajectories of reduced and oxidized F1 molecules. Trajectories of three active single molecules of GH54 driven by ATP hydrolysis both in the reduced (dashed line) and in the oxidized (dotted line) state, respectively. The mean trajectories for each of both states are shown by the solid lines. doi:10.1371/journal.pone.0053754.gUnfolding of Subunit Gamma in Rotary F-ATPasefarther than nine residues away from its C-terminal end. A crosslink at the penultimate residue of the C-terminal end (c285C, MM10) was even without any effect on the activity. In contrast, a cross-link of residues c262C (PP2, middle) or c87C (SW3, bottom) with the stator subunits practically extinguished the hydrolysis activity of F1. Three different lines of evidence support our observation. First, SDS-gels showed a cross-link yield of .85 . Second, bulk phase experiments revealed an activity of cross-linked mutants of at least 26 compared to wild type EF1 that could be restored after rereducing the samples. Third, rotation assay experiments support our conclusions on a single molecule level. Not only did we find single molecules still rotating despite oxidation, but furthermore was the rotational rate reduced by 60 , indicating that rotation was impaired by the cross-link. However, as we observed only a small number of molecules the statistics are only qualitative comparable with bulk phase experiments. What is the reason for this unusual behavior? For steric reasons alone the rotation of subunit c around the engineered disulfide bond can be excluded because that would imply the dragging around the now obliquely oriented residues 24786787 up to the C-terminal end in the before snugly fitting hydrophobic bearing. It is apparent from the crystal structure [1] (Fig. 1) that there is no space within (ab)3 for such a deformation. On the other hand, at the top subunit c is linked to a loop in subunit a that might provide some flexibility to the movement, enabling the tip of subunit c to rotate on a `leash’. However, this is very unlikely for the following reason. Czub and Grubmuller [25] have shown by molecular dynamics ?simulation (MD) that the respective portion of subunit c is at least four times more flexible than the opposing loop in subunit b (and therefore it is likely to be true for subunit a also because of its homology to subunit b), i.e. any torque applied would first result in a deformation of subunit c before subunit a is affected. Disulfide bond cleav.