Interaction of the Protein Retinitis Pigmentosa 2 (RP2) with Langmuir Phospholipid Monolayers

Abstract

A severe form of retinitis pigmentosa is linked to mutations of the 350 residues protein RP2 (retinitis pigmentosa 2). This protein contains a α/β C-terminal domain, a highly hydrophobic β-helix and two acylation sites at the N-terminal. It localizes predominantly to the membrane. However, the parameters responsible for the modulation of RP2 binding to membranes are still largely unknown. The objectives of this research work were to characterize the membrane binding properties of RP2 using Langmuir monolayers. The complete sequence of RP2 was expressed and high purity was achieved. RP2 was injected into the subphase underneath phospholipid monolayers bearing different fatty acyl chains (length and unsaturation) and polar headgroups. RP2 binding was monitored by surface pressure measurements. The injection of RP2 underneath phospholipid monolayers led to an increase in surface pressure which indicates its membrane binding. The surface pressure data demonstrate that the adsorption kinetics of RP2 is independent of pH but is strongly affected by the ionic strength of the subphase as well as by the type of phospholipid fatty acyl chain (length and unsaturation) and headgroup. For example, on the basis of its maximum insertion pressure, RP2 shows a preferential binding onto saturated phospholipid monolayers which is consistent with its postulated localization to rafts. This interaction has been further studied by infrared sprectoscopy. In solution, the amide I band is centered at 1630 cm−1, indicating the presence of the β-helix. In contrast, when injected into the subphase in the absence and presence of a phospholipid monolayer, the amide I band is shifted to longer wavenumbers with components at 1640 and 1655 cm−1. These data thus suggest that RP2 has a preferential orientation in monolayers where the α/β C-terminal domain is oriented towards the monolayer.