Visualizing water molecules in transmembrane proteins using radiolytic labeling methods

Abstract

Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of "ordered" waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.

Figure 1. Radiolytic protein footprinting experiments and data analysis

A) Proteins are exposed to X-rays for different time periods (ms). B) Following exposure the protein is digested with a protease and its fragments are separated by using reverse phase high-performance liquid chromatography coupled to a mass spectrometer. Searches for amino acid modifications such as +16 Da and other specific modifications (see Mechanisms of amino acid – OH· interactions) are performed using programs such as Mascot (Matrix Science) and ProtMapMS (57). C) Identification of the modified species based on its MS/MS spectra. D) The fraction unmodified is calculated by using the selected ion chromatogram peak area (modified in red and unmodified in blue). E) The fraction unmodified is plotted as a function of exposure time and the rate of modification is calculated by using nonlinear regression.

Figure 2. Footprinting of a model GPCR – rhodopsin

Rhodopsin is shown as gray ribbons whereas amino acid residues found to be modified following exposure to X-rays are shown as red sticks. The N- and C-termini are indicated together with the transmembrane region. The rhodopsin chromophore, 11-cis-retinal, is shown in blue near the intradiscal side of the membrane.

Figure 3. Transmembrane proteins and waters detected by crystallography

In panel A rhodopsin (pdb: 1U19) is depicted by gray ribbons and crystallographically determined water molecules are shown as green spheres. The rhodopsin chromophore, 11-cis-retinal, is shown as blue sticks whereas amino acid residues from the transmembrane region in contact with water molecules are shown as red sticks. Panel B shows superposed structures of GPCRs such as squid rhodopsin in magenta, β₁-adrenergic receptor in cyan, β₂-adrenergic receptor in yellow and bovine opsin in red.

Figure 4. Transmembrane water molecules determined by FTIR

Panel A shows rhodopsin as gray ribbons, Asp83 and Gly120 are shown in red whereas the interacting water molecule is shown as a blue sphere. Water molecules determined by X-ray crystallography (pdb 1U19-chain A) are shown as green spheres. Panel B shows a top view of rhodopsin with the same coloring scheme used as in panel A.