Fig 1.
In vivo screens to determine MscL activity changes after post-translational modifications.
(A) The structure of E. coli MscL in its closed state [16], generously provided by Ben Corry, is shown in a side view, with a subunit highlighted for clarity. The approximate location of the lipid membrane headgroups is marked by horizontal tan lines. The domains, in which the study has been divided, are highlighted in an isolated subunit using different colors. (B) A schematic description of the in vivo screens used to study the activity of the MscL cysteine substituted channels before and after post-translational modification with different MTS reagents. In a primary screen, the osmotically fragile strain MJF 455 is osmotically shocked with or without the MTS reagents present during the osmotic down-shock. Channels with reduced sensitivity (Loss of function LOF) or increased sensitive to tension (gain of function GOF), lead to cells with reduce viability in the primary in vivo screens. A secondary screen is done to distinguish between these two phenotypes. The secondary screen consists in MJF367 strain (not osmotically fragile) osmotically shocked with the MTS reagents present in during the osmotic down-shock. In the secondary screens a reduced viability indicates more sensitive channel or GOF. (C) The structure of the sulfhydryl reagents 2-sulfonatoethyl methanethiosulfonate sodium salt (MTSES-), ethyl methanethiosulfonate Bromide (MTSET+), 4-hydroxybenzyl methanethiosulfonate (4-HB-MTS), benzyl methanethiosulfonate (MTSBn), and decyl methanethiosulfonate (decyl-MTS), 2-(Trimethylammonium) are shown.
Fig 2.
Effects of post translational modifications on the N-terminal domain of MscL determined by in vivo screens.
The viability of the osmotically fragile strain MJF455 expressing MscL cysteine substituted mutants from residues S2 to D18 (insert), was measured after an osmotic down-shock. The graphs show the differences in viability between non-treated and post-transnationally modified channels with (A) the hydrophobic MTS reagents MTSBn (blue), decyl-MTS (red) or 4HB-MTS (green) or (B) the negatively charged MTSES- (blue) and positively charged MTSET+ (red). The red grid line indicates a ± 50% change that was used as a threshold for further studies.
Fig 3.
Functional changes by substitutions in the N-terminal domain of MscL, determined by in vivo and patch clamp experiments.
(A) The location of the residues showing changes in viability upon post-translational modifications is highlighted in the closed structure of E. coli MscL. From right to left: a pentameric MscL is shown in a side view followed by a single subunit and a close up of the region and a cytoplasmic view of the pentameric channel. (B) Viability of MJF 367 (mscL-) shocked in the presence of the indicated MTS reagent is shown for each individual MscL mutant. (C) The changes in the pressure threshold required to gate E9C MscL caused by treatment with MTS reagents is graphed as the ratio between before and after modification of the same patch. The red line indicated no change. (D) The changes in the pressure threshold required to gate M12C MscL caused by MTS reagents is graphed as the ratio between before and after modification of the same patch. The red line indicates no change. (E) Representative traces of E9C MscL and M12C before (control) and after treatment with MTSBn. The upper traces correspond to the current and the lower traces the negative pressure applied to the patch.
Fig 4.
Effects of post translational modifications on the TM1 domain of MscL determined by in vivo channel activity.
The viability of the osmotically fragile strain MJF455 expressing MscL cysteine substituted mutants from residues L19 to G46 (insert), was measured after an osmotic down-shock. The graphs show the differences in viability between non-treated and post-transnationally modified channels with (A) the hydrophobic MTS reagents MTSBn (blue), decyl-MTS (red) or 4HB-MTS (green) or (B) the negatively charged MTSES- (blue) and positively charged MTSET+ (red). The red grid line indicates a ± 50% change that was used as a threshold for further studies.
Fig 5.
Functional changes by substitutions in the TM1 domain of MscL, determined by in vivo and patch clamp experiments.
(A) The location of the residues showing changes in viability upon post-translational modifications is highlighted in the closed structure of E. coli MscL. From right to left: a pentameric MscL is shown in cytoplasmic view, a periplasmic view, and a side view where the approximate location of the membrane is shown. A single subunit and a close up of the region with the labeled residues is on the left. (B) Viability of MJF 367 (mscL-) cells, shock in the presence of the indicated hydrophobic MTS reagent is shown for each individual MscL mutant. (C) Viability of MJF 367 osmotically shocked with charged MTS reagents is shown for the indicated cysteine mutants.
Fig 6.
Effects of post translational modifications on the periplasmic loop of MscL determined by in vivo channel activity.
The viability of the osmotically fragile strain MJF455 expressing MscL cysteine substituted mutants from residues L47 to H74 (insert), was measured after an osmotic down-shock. The graphs show the differences in viability between non-treated and post-transnationally modified channels with (A) the hydrophobic MTS reagents MTSBn (blue), decyl-MTS (red) or 4HB-MTS (green) or (B) the negatively charged MTSES- (blue) and positively charged MTSET+ (red). The red grid line indicates a ± 50% change that was used as a threshold for further studies.
Fig 7.
Functional changes by substitutions in the periplasmic loop of MscL, determined by in vivo and patch clamp experiments.
(A) The location of the residues showing changes in viability upon post-translational modifications is highlighted in the closed structure of E. coli MscL. From right to left: a pentameric MscL is shown in cytoplasmic view, a periplasmic view, and a side view where the approximate location of the membrane is shown. A single subunit and a close up of the region with the labeled residues is on the left. (B) Viability of MJF 367 (mscL-) shock in the presence of the indicated MTS reagent is shown for each individual MscL mutant, for hydrophobic or (C) charged MTS reagents.
Fig 8.
Effects of post translational modifications on the TM2 domain of MscL determined by in vivo channel activity.
The viability of the osmotically fragile strain MJF455 expressing MscL cysteine substituted mutants from residues Y75 to N100 (insert), was measured after an osmotic down-shock. The graphs show the differences in viability between non-treated and post-transnationally modified channels with (A) the hydrophobic MTS reagents MTSBn (blue), decyl-MTS (red) or 4HB-MTS (green) or (B) the negatively charged MTSES- (blue) and positively charged MTSET+ (red). The red grid line indicates a ± 50% change that was used as a threshold for further studies.
Fig 9.
Functional changes by substitutions in the TM2 domain of MscL, determined by in vivo and patch clamp experiments.
(A) The location of the residues showing changes in viability upon post-translational modifications is highlighted in the closed structure of E. coli MscL. A pentameric MscL is shown side view where the approximate location of the membrane marked, followed by single subunit with the labeled residues. Cytoplasmic and periplasmic views are also shown below. (B) Viability of MJF 367 (mscL-) cells, shock in the presence of the indicated MTS reagent is shown for each individual MscL, for hydrophobic or C. charged MTS reagents.
Fig 10.
Effects of post translational modifications on the C-terminal domain of MscL determined by in vivo channel activity.
The viability of the osmotically fragile strain MJF455 expressing MscL cysteine substituted mutants from residues K101 to A114 (insert), was measured after an osmotic down-shock. The graphs show the differences in viability between non-treated and post-transnationally modified channels with (A) the hydrophobic MTS reagents MTSBn (blue), decyl-MTS (red) or 4HB-MTS (green) or (B) the negatively charged MTSES- (blue) and positively charged MTSET+ (red). The red grid line indicates a ± 50% change that was used as a threshold for further studies.
Fig 11.
Functional changes by substitutions in the C-terminal domain of MscL, determined by in vivo and patch clamp experiments.
(A) The location of the residues showing changes in viability upon post-translational modifications is highlighted in the closed structure of E. coli MscL. From left to right: a pentameric MscL is shown in a side view where the approximate location of the membrane marked, a single subunit and a close up of the region with the labeled residues followed by a cytoplasmic view. (B) Viability MJF 367 (mscL-) cells shock in the presence of the indicated MTS reagent is shown for each individual MscL mutant. C. The changes in the pressure threshold required to gate R104C, K105C and K106C MscL caused by MTS reagents is graphed as the ratio between before and after modification of the same patch. The red line indicates no change.