Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Abstract

Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII–NpHtrII) was performed. The influence of the polydispersity of NpSRII–NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

Structural insights into 3Fe-4S ferredoxins diversity inM.tuberculosishighlighted by a first redox complex with P450

“…The plot has a maximum value of 1.120 at qR g = 1.786, which is very close to the properties of the peak in the case of ideally globular particles (1.104 at qR g = 3) 70 . A peak shift towards higher qR g values could indicate partial disorder or an elongated protein shape 71,72 , but this was not observed in our data.…”

Structural insights into 3Fe–4S ferredoxins diversity in M. tuberculosis highlighted by a first redox complex with P450

“…The plot has a maximum value of 1.120 at qR g = 1.786, which is very close to the properties of the peak in the case of ideally globular particles (1.104 at qR g = √3) ( Burger et al, 2016 ). A peak shift toward higher qR g values could indicate partial disorder or an elongated protein shape ( Ryzhykau et al, 2021a ; Ryzhykau et al, 2021b ), but this was not observed in our data.…”

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review

“…Therefore, the direct application of these classical ab initio approaches to the SAXS data obtained for MPs may provide misleading results. For example, the presence of detergents or lipids bound to MPs results in the appearance of scattering intensity maxima at q ≈ 0.1–0.2 Å −1 (demonstrated for the sensory rhodopsin II/transducer complex from Natronomonas pharaonis [ 158 , 159 ]). In SMA-solubilized lipodiscs, the polymer may be located not only at the rim of the lipodisc, but also on its surface, affecting the scattering intensity [ 65 ].…”