# Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core

## TL;DR

This paper explores how Aquaporin 1 folds in the membrane, showing that helix 3 can shift out of the membrane core during the process.

## Contribution

The study proposes a novel mechanism for helix 3 reorientation in AQP1 folding, supported by experimental and computational evidence.

## Key findings

- The loop preceding helix 3 can insert into the membrane, supporting helix 3's shift.
- Hydrophobicity and simulations suggest a low barrier for helix 3 to slide out of the membrane core.
- This shift initiates a rearrangement leading to the final six-helical conformation of AQP1.

## Abstract

The folding of most integral membrane proteins follows a two-step process: Initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four-helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step this intermediate is folded into a six-helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: First, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3 and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD-simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process