Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T1 NMR Relaxation

Abstract

A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent N NMR relaxation times in the rotating frame, or T, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sinθ, where θ is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D = 8.0 × 10 s was inferred from linear regression of the experimental T data even without any spectroscopic assignment. Alternatively, a close value of D = 7.7 × 10 s was obtained by fitting the T relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.

Rotational Diffusion of Membrane Proteins in Crowded Membranes

“…Notably, the values from two independent STEPR experiments for rhodopsin in model membranes, (0.3–1) × 10 5 rad 2 /s, , agree well with the aforementioned values obtained from living cells using fluorescence . Additionally, NMR spin relaxation experiments can detect the rotational dynamics of membrane proteins, but additional models are needed to determine the rotational diffusion coefficients around the axis normal to the membrane plane because the detected chemical bonds may not rotate in parallel to the membrane plane. ,, …”

“…24 Additionally, NMR spin relaxation experiments can detect rotational dynamics of membrane proteins, but additional models are needed to determine rotational diffusion coefficients around membrane normal because the detected chemical bonds may not rotate in parallel to the membrane plane. 14,26,27 Computationally, the rotational diffusion of synthetic inclusions in lipid bilayers was evaluated first using dissipative particle dynamics simulations. 28 They revealed that the SD description agrees well with simulation data for inclusions whose size differs by multiple orders of magnitude.…”

“…The notation Pr[1][2][3][4][5][6][7] refers to all seven proteins and D[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] to all the 15 disks. single alpha helix 27. Rotational dynamics of proteins in atomistic MD simulations in solution was previously compared directly with NMR spin relaxation experiments detecting motions of backbone N-H bonds, 38 but this is not feasible here because atoms are not explicitly modelled in coarse grained simulations.…”

Finite-Size-Corrected Rotational Diffusion Coefficients of Membrane Proteins and Carbon Nanotubes from Molecular Dynamics Simulations

“…9,11,12 In contrast to translational diffusion, the finite-size behavior of rotational diffusion in the membrane has remained largely unstudied, even though corrections may be required for meaningful comparisons to experiment. 13,14…”

Evaluation of the Rotating-Frame Relaxation (T1ρ) Filter and Its Application in Metabolomics as an Alternative to the Transverse Relaxation (T2) Filter

“…Conversely, it is widely used in solid-state NMR and magnetic resonance imaging (MRI) − to improve the contrasts in the images, and several developments have been undertaken to mitigate the adverse effects of spin-locking such as adiabatic rf pulses , or phase cycling methods for simultaneous B 1 rf and B 0 field inhomogeneity compensation. , In liquid state, T 1ρ filtering is usually used in the nuclear Overhauser effect and saturation transfer difference experiments in protein–ligand interaction studies for the elimination of protein signals . There are many potential fields of application reported in the literature, particularly in the study of complex and heterogeneous biological systems in solution and HR-MAS NMR such as lipid bilayers, nanodiscs, membrane proteins, and bicelles. − For example, the use of laboratory and rotating-frame experiments such as INEPT and CP is well described for HR-MAS and solid-state NMR for the characterization of the membrane topology and dynamic properties of full-length rabbit cyt-b5 in a membrane environment and more recently, in the in situ determination of the molecular structure of cartilage . In HR-MAS spectroscopy, it has been used in several systems but has never been performed and assessed in the context of metabolomics.…”