X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins

Abstract

Membrane proteins are distinguished from soluble proteins by their insertion into biological membranes. This insertion is achieved via a noticeable arrangement of hydrophobic amino acids that are exposed at the surface of the protein, and renders the interaction with the aliphatic tails of lipids more energetically favorable. This important difference between these two categories of proteins is the source of the need for a specific handling of membrane proteins, which transpired in the creation of new tools for their recombinant expression, purification and even crystallization. Following this line, we show here that crystals of membrane proteins display systematically higher diffraction anisotropy than those of soluble proteins. This phenomenon dramatically hampers structure solution and refinement, and has a strong impact on the quality of electron-density maps. A farther search for origins of this phenomenon showed that the type of crystallization, and thus the crystal packing, has no impact on anisotropy, nor does the nature or function of the membrane protein. Membrane proteins fully embedded within the membrane display equal anisotropy compared to the ones with extra membranous domains or fusions with soluble proteins. Overall, these results overturn common beliefs and call for a specific handling of their diffraction data.

Figure 1

Probability distribution fonction of anisotropy. Soluble proteins and membrane proteins are depicted as blue and red bars respectively. (a) Anisotropy calculated on amplitudes (F). (b) Anisotropy calculated on intensities (I). The distribution fits (Weibull distributions) are shown as black line for soluble proteins and white line for membrane proteins.

Figure 2

Anisotropy as a function of resolution. Values of anisotropy were calculated on amplitudes (a) or intensities (b). Soluble proteins are depicted in blue, membrane proteins in red. Each dot represents an entry (soluble proteins: 74,928 amplitudes and 22,985 intensities data; membrane proteins: 1,414 amplitudes and 489 intensities data). The bold dots represent the 25^th, 50^th, 75^th or 95^th percentile per resolution bin, fitted with a power law. For soluble proteins, the 99^th percentile is also represented. The area under the fit has been filled with transparent colors according to the percentile.

Figure 3

Comparaison of membrane proteins crystallized in detergents vs LCP. (a) AnisoB: T-test two-tailed p-value = 0.0009; M-W two-tailed p-value = 0.9733. Inset: comparison of anisotropy for structures between 2.5 and 3 Å resolution. Student’s T-test with Welch correction: two-tailed p-value = 0.22; M-W two-tailed p-value = 0.85. (b) Solvent Content: T-test two-tailed p-value < 0.0001; M-W two-tailed p-value < 0.0001. (c) Crystal contacts ratio: T-test two-tailed p-value = 0.0274; M-W two-tailed p-value < 0.0001. (d) Resolution: T-test two-tailed p-value < 0.0001; M-W two-tailed p-value = 0.0007.

Figure 4

Comparaison membrane proteins fully embeded compared to those having extra-membranous-domains. (a) AnisoB: T-test with Welch correction two-tailed p-value = 0.0005; mean embedded = 22.3 Ų, mean extra-membranous-domains = 27.4 Ų; M-W two-tailed p-value = 0.0003. (b) AnisoB for structures between 2.8 and 3 Å resolution: T-test two-tailed p-value > 0.99, mean = 29.44 Ų; M-W two-tailed p-value > 0.99. (c) Crystal contacts: Student’s T-test with Welch correction: two-tailed p-value < 0.0001, mean embedded = 0.089, mean extra-MB-domain = 0.057; M-W two-tailed p-value < 0.0001. (d) Resolution: Student’s T-test with Welch correction: two-tailed p-value < 0.0001, mean embedded = 2.67 Å, mean extra-MB-domain = 2.99 Å; M-W two-tailed p-value < 0.0001.