Dataset Information

Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D ²H-¹³C-¹³C Solid-State NMR Spectroscopy.

ABSTRACT: (2)H quadrupolar line shapes deliver rich information about protein dynamics. A newly designed 3D (2)H-(13)C-(13)C solid-state NMR magic angle spinning (MAS) experiment is presented and demonstrated on the microcrystalline ?1 immunoglobulin binding domain of protein G (GB1). The implementation of (2)H-(13)C adiabatic rotor-echo-short-pulse-irradiation cross-polarization (RESPIRATION CP) ensures the accuracy of the extracted line shapes and provides enhanced sensitivity relative to conventional CP methods. The 3D (2)H-(13)C-(13)C spectrum reveals (2)H line shapes for 140 resolved aliphatic deuterium sites. Motional-averaged (2)H quadrupolar parameters obtained from the line-shape fitting identify side-chain motions. Restricted side-chain dynamics are observed for a number of polar residues including K13, D22, E27, K31, D36, N37, D46, D47, K50, and E56, which we attribute to the effects of salt bridges and hydrogen bonds. In contrast, we observe significantly enhanced side-chain flexibility for Q2, K4, K10, E15, E19, N35, N40, and E42, due to solvent exposure and low packing density. T11, T16, and T17 side chains exhibit motions with larger amplitudes than other Thr residues due to solvent interactions. The side chains of L5, V54, and V29 are highly rigid because they are packed in the core of the protein. High correlations were demonstrated between GB1 side-chain dynamics and its biological function. Large-amplitude side-chain motions are observed for regions contacting and interacting with immunoglobulin G (IgG). In contrast, rigid side chains are primarily found for residues in the structural core of the protein that are absent from protein binding and interactions.

SUBMITTER: Shi X

PROVIDER: S-EPMC4819898 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Publications

Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D ²H-¹³C-¹³C Solid-State NMR Spectroscopy.

Shi Xiangyan X Rienstra Chad M CM

Journal of the American Chemical Society 20160318 12

(2)H quadrupolar line shapes deliver rich information about protein dynamics. A newly designed 3D (2)H-(13)C-(13)C solid-state NMR magic angle spinning (MAS) experiment is presented and demonstrated on the microcrystalline β1 immunoglobulin binding domain of protein G (GB1). The implementation of (2)H-(13)C adiabatic rotor-echo-short-pulse-irradiation cross-polarization (RESPIRATION CP) ensures the accuracy of the extracted line shapes and provides enhanced sensitivity relative to conventional C ...[more]

PMID: 26849428

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Project description:Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state (2)H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific (2)H labels have been introduced into the methyl groups of retinal and solid-state (2)H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent (2)H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the beta-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the beta4 strand of the E2 loop and the side chains of Glu(122) and Trp(265) within the binding pocket. The solid-state (2)H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

Dataset Information

Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D ²H-¹³C-¹³C Solid-State NMR Spectroscopy.

Publications

Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D ²H-¹³C-¹³C Solid-State NMR Spectroscopy.

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