ESTRATEGIAS PARA GARANTIZAR EL RIGOR METODOLÓGICO

con LG. In the first case, we assume that FE for the open LG is far lower than that of the closed LG, such that the LG is always open. In the second case, we assume that the FE for LG opening remains fixed at a value that favors the closed LG conformation, regard- less of the nascent protein conformation. Unlike the approach employed in chapter 4 (and described in the previous section), numerical tests reveal that both of these alternative de- scriptions of the LG energetics lead to qualitatively incorrect results.

Figure C.5: Testing the effect of LG motions on protein topogenesis and stop-transfer ef- ficiency. We consider the alternative description of the translocon LG in which the LG is always left open. (A)The figure plots Type II integration fraction as a function of MDL. The red data set corresponds to the protein topogenesis results presented for the RL4E SP

sequence in Figure 4.3A of chapter 4. The blue data set is obtained using the same protein sequences and employs the alternative description in which the LG is open at all times in the simulations. (B)The figure plots stop-transfer efficiency as a function of H-domain hy- drophobicity. The black dashed line is the sigmoidal fit to the data presented in Figure 4.5A of chapter 4. The blue data set is obtained using the same protein sequences and employs the assumption that the LG is open at all times in the simulations.

C.1.6.1 Assumption that the LG is Always Open

Figure C.5A shows that neglecting LG opening/closing significantly impacts the calculated results for nascent protein topogenesis. The red data set corresponds to the protein topo- genesis results presented for the RL4E SP sequence in Figure 4.3A of chapter 4. The blue

data set is obtained using the same protein sequences and assuming that the LG is open at all times in the simulations. The neglect of LG opening/closing leads to the complete loss of Type III membrane integration in this case. In the absence of the slow timescale for LG opening, the SP readily adopts the thermodynamically favorable Type II orientation.

Figure C.5B shows similarly discouraging results for stop-transfer efficiency. The dashed line in the figure corresponds to the stop-transfer efficiency results reported in Fig- ure 4.5A of chapter 4. The blue data set is obtained using the same protein sequences and

Figure C.6: Translocation mechanism observed in simulations for which the LG is kept open. Although the open LG enables the H-domain to partition from the channel interior to the membrane interior (a,b), diffusion of the H-domain away from the translocon to yield the membrane integration product is hindered by the attraction of the hydrophilic C-terminal domain for the channel interior (c). Without allowing for LG closing, the C-terminus effec- tively tethers the H-domain to the ribosome until secretion of the C-terminal tail occurs (d), exclusively leading to the secretion product (e). For very hydrophobic H-domain, the final transition fromd toedoes not occur on the timescale of the simulation performed here, yet secretion of the nascent-protein mature domain is complete.

employs the assumption that the LG is open at all times in the simulations. The alterna- tive treatment of the LG leads to complete loss of stop-transfer efficiency; all trajectories lead to the translocation of the C-terminal domain. The mechanistic basis for this result is illustrated in Figure C.6. Although the open LG enables the H-domain to partition from the channel interior to the membrane interior, diffusion of the H-domain away from the translocon to yield the membrane integration product is hindered by the attraction of the hydrophilic C-terminal domain for the channel interior. Without allowing for LG clos- ing, the C-terminus effectively tethers the H-domain to the ribosome until secretion of the C-terminal tail occurs, exclusively leading to the secretion product.

C.1.6.2 Assumption that the FE for LG Opening is Unaffected by the Nascent Pro- tein

We now consider the alternative description in which the FE for LG opening is indepen- dent of the nascent protein contents of the channel. Specifically, we perform simulations in which the relative rates for LG opening and closing (equations (4.11) and (4.12)) corre-

spond to∆Gtot=∆Gempty, regardless of the nascent protein configuration; we employ the

same value for the opening/closing attempt timescale,τ=500 ns, as is used in chapter 4. Figure C.7A shows the effect of the alternative description on nascent protein topogen- esis. The red data set corresponds to the protein topogenesis results presented for the RL4E

SP sequence in Figure 4.3A of chapter 4. The blue data set is obtained using the same protein sequences and using the alternative description for the LG energetics. This leads to almost complete loss of Type II integration for all MDL. Without the role of hydropho- bic SP residues in stabilizing open LG configurations, closed LG configurations dominate. Both the direct and flipping pathways for Type II integration are thus eliminated. Type III integration survives by having the SP enter directly into the membrane interior from the ribosome enclosure, without passing through the translocon channel interior. These results are clearly inconsistent with the experimental observation of Type II membrane integration. Figure C.7B illustrates the effect of the alternative description on the stop-transfer sim- ulations. The dashed line in the figure corresponds to the stop-transfer results reported in Figure 4.5A of chapter 4; the blue data set employs the alternative description, which neglects the effect of the nascent protein on the FE for LG opening. The alternative descrip- tion leads to a significant shift toward reduced membrane integration, although sigmoidal behavior of the stop-transfer efficiency as a function of H-domain hydrophobicity is still observed. As for the topogenesis simulations, closed LG conformations dominate in the alternative description, due to the neglect of the role of H-domain residues in stabilizing the open configurations of the LG. The rapid equilibration of the CG trajectories between statesbandc∗(Figure 4.4) is thus shifted toward stateb, which favors the subsequent for-

Figure C.7: Testing the effect of LG motions on protein topogenesis and stop-transfer ef- ficiency. We consider the alternative description of the translocon LG in which the FE for opening the LG is assumed to be unaffected by the nascent-protein chain. (A)The figure plots Type II integration fraction as a function of MDL. The red data set corresponds to the protein topogenesis results presented for the RL4E SP sequence in Figure 4.3A of chapter 4.

The blue data set is obtained using the same protein sequences and employs the alternative description, which neglects the effect of the nascent protein on the FE for LG opening. (B)

The figure plots stop-transfer efficiency as a function of H-domain hydrophobicity. The black dashed line is the sigmoidal fit to the data presented in Figure 4.5A of chapter 4. The blue data set is obtained using the same protein sequences and employs the alternative description, which neglects the effect of the nascent protein on the FE for LG opening.

mation of the secretion product (as is described in connection with Figure 4.5 in chapter 4 or inAnalytical Model for TM Partitioning).