Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR.

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.2 GHz proton frequency) and ultrafast MAS spinning (<100 kHz) promise to accelerate acquisition, while reducing sample requirement, both of which are critical to membrane protein studies. Here, we review recent advances in ssNMR methodology used for structure determination of membrane proteins in native and mimetic environments, as well as the study of protein functions such as protein dynamics, and interactions with ligands, lipids and cholesterol.

Proton Detected Solid-State NMR of Membrane Proteins at 28 Tesla (1.2 GHz) and 100 kHz Magic-Angle Spinning.

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1200 MHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.

Macroscalar Helices Co-Assembled from Chirality-Transferring Temperature-Responsive Carbohydrate-Based Bolaamphiphiles and 1,4-Benzenediboronic Acid.

We present the first example of macroscalar helices co-assembled from temperature-responsive carbohydrate-based bolaamphiphiles (CHO-Bolas) and 1,4-benzenediboronic acid (BDBA). The CHO-Bolas contained hydrophilic glucose or mannose moieties and a hydrophobic coumarin dimer. They showed temperature-responsive reversible micelle-to-vesicle transition (MVT) in aqueous solutions. After the binding of carbohydrate moieties with boronic acids of BDBA in their alkaline solutions, right-handed helices were formed via the temperature-driven chirality transfer of d-glucose or d-mannose from the molecular to supramolecular level. These helices were co-assembled by unreacted BDBA, boronate esters (B-O-C bonds) between CHO-Bolas and BDBA, as well as boroxine anhydrides (B-O-B bonds) of self-condensed BDBA. After heating at 300 °C under nitrogen, the helices displayed excellent morphological stability. Moreover, they emitted bright blue luminescence caused by strong self-condensation of BDBA and decomposition of coumarin dimers.

Transferred-Rotational-Echo Double Resonance.

Internuclear distance determination is the foundation for NMR-based structure calculation. However, high-precision distance measurement is a laborious process requiring lengthy data acquisitions due to the large set of multidimensional spectra needed at different mixing times. This prevents application to large or challenging molecular systems. Here, we present a new approach, transferred-rotational-echo double resonance (TREDOR), a heteronuclear transfer method in which we simultaneously detect both starting and transferred signals in a single spectrum. This co-acquisition is used to compensate for coherence decay, resulting in accurate and precise distance determination by a single parameter fit using a single spectrum recorded at an ideal mixing time. We showcase TREDOR with the microcrystalline SH3 protein using 3D spectra to resolve resonances. By combining the measured N-C and H-C distances, we calculate the structure of SH3, which converges to the correct fold, with a root-mean-square deviation of 2.1 Å compared to a reference X-ray structure. The TREDOR data used in the structure calculation were acquired in only 4 days on a 600 MHz instrument. This is achieved due to the more than 2-fold time saving afforded by co-acquisition of additional information and demonstrates TREDOR as a fast and straightforward method for determining structures via magic-angle spinning NMR.