Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors

Abstract

Thermostabilization of a membrane proteins, especially G-protein-coupled receptors (GPCRs), is often necessary for biochemical applications and pharmaceutical studies involving structure-based drug design. Here we review our theoretical, physicsbased method for identifying thermostabilizing amino acid mutations. Its novel aspects are the following: The entropic effect originating from the translational displacement of hydrocarbon groups within the lipid bilayer is treated as a pivotal factor; a reliable measure of thermostability is introduced and a mutation which enlarges the measure to a significant extent is chosen; and all the possible mutations can be examined with moderate computational effort. It was shown that mutating the residue at a position of N BW = 3.39 (N BW is the Ballesteros-Weinstein number) to Arg or Lys leads to the stabilization of significantly many different GPCRs of class A in the inactive state. Up to now, we have been successful in stabilizing several GPCRs and newly solving three-dimensional structures for the muscarinic acetylcholine receptor 2 (M2R), prostaglandin E receptor 4 (EP4), and serotonin 2A receptor (5-HT 2A R) using X-ray crystallography. The subjects to be pursued in future studies are also discussed.

Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations

“…The thermostabilization of a membrane protein using amino-acid mutations is not straightforward even for a rather unstable protein (e.g., a G-protein coupled receptor (GPCR)), to say nothing for an intrinsically thermostable protein. We first developed a theory for evaluating the structural stability of a GPCR − and then extended it to a rhodopsin. − Recently, we developed method 1 suited to the identification of stabilizing mutations for rhodopsins with high values of T m . We believe that the extended version and method 1 as well as the methodology proposed in the present article is applicable to GPCRs and rhodopsins with a wide range of T m -values, which should be verified in future investigations.…”

“…The three-dimensional (3D) structure, one of the most important characteristics, then becomes difficult to solve by X-ray crystallography. Amino-acid mutations provide the most promising means of thermostabilization which should lead to the stabilization against diverse perturbing factors other than the heat as well. − First, we developed a theory based on statistical thermodynamics for identifying thermostabilizing mutations for a human GPCR, which considers only the TM portion. − It emphasizes the entropic effect originating from the translational displacement of the hydrocarbon groups referred to above. This development led to the new solution of 3D structures of human prostaglandin E receptor EP4, M2 muscarinic receptor, and serotonin 2A receptor (5-HT 2A R) .…”

Biophysical Reviews: 2020—looking back, going forward

“…The bulk of the Special Issue was composed of numerous review articles (forty-five) chosen from the nearly thirty separate sections of the BSJ’s national society meeting. A short selection of these (many) interesting review articles includes those dealing with the biophysics of chromatin (Ashwin et al 2020 ; Kumar and Kono 2020 ); optogenetics (Kandori 2020 ); computational structure prediction (Kinoshita and Hayashi 2020 ; Leitner and Yamamoto 2020 ; Tsuchiya and Tomii 2020 ); physical biochemistry (Tsumoto et al 2020 ); molecular motors (Li and Toyabe 2020 ; Loutschko and Flechsig 2020 ; Noji et al 2020 ); membrane protein interaction (Moghal et al 2020 ); novel scattering, structural and imaging techniques (Nakasako et al 2020 ; Uchihashi and Ganser 2020 ; Yamaoki et al 2020 ; Yokoyama et al 2020 ); cellular biophysics (Okazaki et al 2020 ; Yasuda 2020 ); biophysical thermochemistry (Fukuyama and Maeda 2020 ; Suzuki and Plakhotnik 2020 ); and biophysical theory (Leitner and Yamamoto 2020 ; Uda 2020 ).…”

Sample preparation of membrane proteins using anti-His tag antibody

“…The difficulty in producing target molecules has been a bottleneck in drug discovery research. Over the years, we have been conducting fundamental research focused on membrane proteins and have developed a technology to predict heat-resistant mutants of membrane proteins using our unique theoretical calculations [1,2]. A significant advantage of our calculation method is its very short computational time, as it handles entropy effects originating from the translational movement of hydrocarbon chains in lipid molecules from a statistical mechanics standpoint.…”