Microscale NMR Screening of New Detergents for Membrane Protein Structural Biology

Abstract

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly (2)H, (15)N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ (15)N, (1)H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

“…[19] Here, we explored effects of the detergent-free environment in 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) nanodiscs on the structure and dynamics of OmpA, using newly recorded corresponding measurements with Fos-10-reconstituted OmpA and literature data on studies with different detergents [8–11] as a reference. To this end, micro-coil NMR diffusion measurements [20–22,28] were used, and enzymatic modification of OmpA in the nanodiscs has been implemented.…”

In vitro prediction of human intestinal absorption and blood–brain barrier partitioning: development of a lipid analog for micellar liquid chromatography

“…Our synthesis route was slightly modified from a previously reported procedure by Zhang et al [31]. A mixture of cetyl alcohol (100 mg, 0.41 mmol) and Et 3 N (288 μL, 2.06 mmol) in anhydrous toluene (8.25 mL, 77.90 mmol) was cooled to 0°C.…”

“…Several methods exist for the estimation of the CMC of surfactants. These methods include the use of fluorimetric titrations of ANS or rhodamine 6G [31,61,62] and the variation of surface tension [33,[63][64][65].…”

“…Thus far, they have not been used as surfactants in MLC, probably as a consequence of cost and solubility issues of many natural phospholipids. However, various synthesis routes have been described for phospholipids [24][25][26], lysophospholipids [27], and alkylphosphocholines [28][29][30][31][32][33][34]. Miltefosine, an alkylphosphocholine, is an antitumor drug [25,29,32,35] which has also been tested for the oral treatment of visceral leishmaniasis [26,36] and for its antifungal and antimicrobial activity [30,34].…”

NMR characterization of foldedness for the production of E3 RING domains

“…The spectral dispersion, as well as the signal intensities have been taken as indications for foldedness, and thus a comparison of spectra of different deletion constructs can identify the parts that belong structured elements both using 1D Rehm et al, 2002;Reich et al, 2006) and 2D 15 N-1 H HSQC NMR spectroscopy (Folkers et al, 2004;Galvao-Botton et al, 2003;Yee et al, 2002). Improved sensitivity from the use of microcoil NMR probes, cryogenically-cooled NMR probes, or the combination of both enables NMR screening at high throughput in relatively small volumes Rossi et al, 2010;Zhang et al, 2008) and this process can be partially automated (Jensen et al, 2010). Improvement of protein solubility and long-term stability can be achieved by adding additives and can be simply evaluated from a single or a time series of NMR spectra (Golovanov et al, 2004), or by attaching small fusion proteins (Huth et al, 1997;Zhou and Wagner, 2010;Zhou et al, 2001).…”