Solid-State¹⁷O NMR as a Probe for Structural Studies of Proteins in Biomembranes

Abstract

A key experimental challenge to understand conformational details of membrane proteins is to provide unambiguous atomic-scale information about the molecular bonding arrangement and any changes that occur upon receptor activation. This demands the development of experimental probe techniques to deliver this information of biological and pharmaceutical importance. Solid-state NMR is a nonperturbing approach which can be used to study ligandprotein interactions where molecular size is not limiting and crystallinity is not a requirement. 1 As a first step in addressing this challenge by exploiting 17 O NMR in biomembrane applications, we report here the 17 O solid-state NMR spectra at high field of an 17 O selectively labeled transmembrane peptide in a biomimetic environment.Oxygen plays a key role in the molecular conformation of biopolymers. Among the important information that can be obtained from 17 O, the only NMR-active oxygen isotope, is the isotropic chemical shift (δ iso ), the quadrupolar coupling constant (C Q ), and the asymmetry parameter (η). These parameters are extremely sensitive to the electronic distribution around the nucleus; more specifically, they are sensitive to its protonation state 2 and its involvement in hydrogen bonds. Furthermore, they contain structural information, 2-5 and several methods for determining internuclear distances between oxygen and other nuclei have been developed. 2,6,7 This suggests that oxygen could play a central role in biological NMR studies. However, 17 O has a low natural abundance (0.037%) and a spin (I ) 5 / 2 ) with a corresponding quadrupole interaction that is manifested as significantly broadened signals in NMR spectra. Consequently, 17 O solid-state NMR studies are still relatively uncommon, and selective labeling is essential. Despite these difficulties, in recent years, with the advent of higher magnetic fields and techniques for improving resolution beyond magic angle spinning experiments (MAS), there has been a significant increase in 17 O NMR reports from inorganic materials, such as glasses and zeolites. 8,9 There has been much less 17 O NMR reported from organic materials since 17 O presents even more of a challenge due to the larger quadrupole interaction and, hence, larger line widths. 19 Recent reports of 17 O NMR from biologically relevant materials have included inorganic molecules interacting with hemeproteins, 10 polypeptides, 11,12 amino acids, 2 and nucleic acid bases. 13 Here, we report the first example of 17 O NMR spectra from a selectively labeled transmembrane peptide, 17 O-[Ala12]-WALP23, as a lyophilized powder and incorporated in hydrated vesicles, opening up new possibilities for applications of 17 O solid-state NMR on real biological systems. WALP23 is a synthetic peptide which represents a consensus sequence for transmembrane protein segments. 14 This hydrophobic peptide forms well-defined and wellcharacterized transmembrane R-helices 14 and has special relevance

Solid‐State 17O NMR Spectroscopy of Large Protein–Ligand Complexes

“…Later, Oldfield and co-workers [4] successfully obtained solid-state 17 O NMR spectra for [C 17 O]myoglobin (16.7 kDa per ligand) under conditions of magic-angle spinning (MAS) at 11.7 T. Several groups have since reported solid-state 17 O NMR spectroscopic studies of membrane-bound peptides. [5][6][7] Herein we report the first comprehensive solid-state 17 17 O spin-lattice relaxation times (T 1 ) in these solid protein-ligand complexes are typically on the order of several milliseconds; therefore, very rapid data collection is possible. Furthermore, we found that several sensitivity-enhancement methods uniquely suited for halfinteger quadrupolar nuclei, such as double-frequency sweep (DFS), [8] rotor-assisted population transfer (RAPT), [9] and hyperbolic secant (HS) pulses, [10] can be used to obtain highquality solid-state 17 O NMR spectra for large protein-ligand complexes.…”

Structural Studies on the Hydration of l-Glutamic Acid in Solution

“…This labeling was used because the COOgroups have been shown to be distinguishable from one another by solid-state NMR measurements. 16 Finally the COO -groups and the NH 3 + group on each glutamic acid molecule were allowed to rotate in the model about the appropriate C-C or C-N bond.…”

Sensitivity Enhancement and Heteronuclear Distance Measurements in Biological 17O Solid-State NMR

“…Solid-state NMR spectroscopy of 17 O is a powerful tool to study hydrogen bonds since the 17 O chemical shift and quadrupolar coupling are claimed to be very sensitive to the strength and geometry of the hydrogen bonds. In recent years, several groups have demonstrated the feasibility of 17 O NMR experiments in small organic molecules, [3][4][5][6] nucleic acid basis, 7 amino acids, 3,6,[8][9][10] polypeptides, 11,12 and proteins, 13,14 showing a correlation between hydrogen bonding and 17 O chemical shifts and quadrupolar couplings. 17 O is a quadrupolar nucleus (S ) 5 / 2 ) with a low sensitivity and a low natural abundance.…”