Many methods are available for prediction of topology of transmembrane helices, this being one of the success stories of protein structure prediction with accuracies over 90%. However, there are still areas where there is disagreement in some areas about the partitioning between the states of dissolved in water and positioned across a lipid bilayer. Complications arise because there are so many methods of measuring the thermodynamics of this transition – experimental and theoretical, in vivo and in vitro. It is uncertain what difference the translocon makes to the energetics of insertion – is the topology and conformation of a membrane protein the global thermodynamic minimum or just a kinetic product?

This paper uses three approaches to measure partitioning to test the agreement between different methods. The authors aim to reconcile differences calculated so far for insertion of an arginine residue into the membrane (ranging from +2 to +15 kcal/mol). This is an important question, because many transmembrane helices are only marginally hydrophobic and it is not known how and when they insert in the folding process. Arginine is chosen here because the pKa of 12.5 of the side chain is very high so it will not deprotonate in the centre of a bilayer and complications of protonation and deprotonation do not need to be considered. The same peptide is used for each method, of the form L_nRL_n, and the ratio between the interface and transmembrane states is used to calculate estimates of ΔG. In order to make sure that there were helices with a ΔG close to zero for accurate estimates, they used a range of values of n from 5-8.

The first method was an insertion assay using reconstituted microsomes, where this helix was inserted into the luminal domain of LepB. A glycosylation site was added at each end of the helix, but glycosylation takes place only on sites inside microsomes. Helices inserted into the membrane are only glycosylated once, whereas secreted helices are glycosylated twice and those which did not go through the translocon are not glycosylated. SDS-PAGE can separate these states by mass, and the ratio between single and double glycosylation gives the partitioning between inserted and interface helices out of those which entered the translocon. As expected, the trend is for longer helices with more leucine to favour the transmembrane state.

Adapted from Figure 4a: The helix, H, either passes through the translocon into the lumen (“S”) resulting in two glycosylations (green pentagons), or is inserted (TM) resulting in one glycosylation.

The second method was also experimental: oriented synchrotron radiation circular dichroism (ORSCD). Here they used just the peptide with one glycine at each end, as this would be able to equilibrate between the two states quickly. Theoretical spectra can be calculated for a helix , and therefore the ratio in which they must be combined to give the measured spectrum for a given peptide gives the ratio of transmembrane and interface states present.

Figure 2b: TM and IP are the theoretical spectra for the transmembrane and interface states, and the peptides fall somewhere in between.

Finally, the authors present 4 μs molecular dynamics simulations of the same peptides at 140°C, so that equilibration between the two states would be fast. The extended peptide at the start of the simulation quickly associates with the membrane and adopts a helical conformation. An important observation to note is that the transmembrane state is in fact at around 30° to the membrane normal, to allow the charged guanidinium group of the arginine to “snorkel” up to interact with charged phosphate groups of the lipids. Therefore this state is defined as transmembrane, in contrast to the OSRCD experiments where the theoretical TM spectrum was calculated for a perpendicular helix. This may be a source of some inaccuracy in the propensities calculated from OSRCD.

Figure 2c: Equilibration in the simulation for the L₇RL₇ peptide. Transmembrane and interface states are seen in the partitioning and equilibration phases after the helix has formed.

Figure 3c: As the simulations run, the proportion of helices in the transmembrane state (P_TM) converges to a different value for each peptide.

Overall, the ΔG calculated experimental and molecular dynamics (MD) simulations agree very well. In fact, they agree better than those from previous studies of a similar format looking at polyleucine helices, where there was a consistent offset of 2 kcal/mol between the experiment and simulation derived values. The authors are unable to explain why the agreement for this study is better, but they indicate that it is unlikely to be related to any stabilisation by dimerisation in the experimental results, as a 4 μs MD simulation of two helices did not show them forming stable interactions. The calculated difference in insertion energy (ΔΔG) on replacing a leucine with argnine is therefore calculated to be +2.4-4.3 kcal/mol by experiment and +5.4-6.8 by simulation, depending on the length of the peptide (it is a more costly substitution for longer peptides as the charge is buried deeper). The difference between the experimental and simulation results is accounted for by their disagreement in the polyleucine study.

We thought this paper was a great example of experimental design, where the system was carefully chosen so that different experimental and theoretical approaches would be directly comparable. The outcome is good agreement between the methods, demonstrating that the vastly different values recorded previously seem to be because very different questions were being asked.