Macroscopic Orientation of Natural and Model Membranes for Structural Studies

Abstract

O ne a p p ro a ch for o b ta in in g h ig h -r eso lu tio n s tr u c tu ra l an d fu n ctio n a l in fo rm a tio n for b iom em b ran es an d th e ir p ro tein s is b y s ta tic so lid -sta te NMR o f o ri e n te d sy stem s. H ere, a g e n e r a l p ro ced u re to a lig n fu lly fu n c tio n a l b io lo g ica l m em b ra n es co n ta in in g large m em b ran e p ro tein s (Mr >30,000) is d escrib ed . T he m eth od , b a sed on th e iso p o te n tia l sp in -d ry u ltra ce n tr ifu g a tio n tech n iq u e , r e lie s on th e cen trifu g a tio n o f m em b ran e fra g m en ts on to a su p p o rt w ith sim u lta n e ous, or su b seq u en t, p a r tia l e v a p o r a tio n o f th e so lv e n t w h ic h a id s a lig n m en t. atu ral m em b ra n es w ith th e red c e ll a n io n ex ch a n g e tran sp ort p r o te in in ery th ro cy tes, b an d 3, an d th e n ico tin ic a c e ty lc h o lin e receptor. O 1997 Academic Press The elucidation of the structural and functional orga nization of biological membranes by various biophysi cal techniques is one of the important questions being addressed in structural biology. The complexity of the systems, however, their supramolecular structure, and their dynamic properties make direct studies particu larly difficult. Specifically, membrane proteins present extraordinary technical difficulties to the most com monly used methods in structural biology, such as crys tallography (1) and solution NMR spectroscopy (2). In 1 To whom correspondence should be addressed, Fax; 01865/275-234 or -259. E-mail: awatts@bioch.ox.ac.uk. 132recent years solid-state NMR spectroscopy (3, 4) on macroscopically oriented lipid bilayers has rapidly emerged as an alternative approach to elucidate struc tural and functional features of membrane-bound pep tides and proteins. In such aligned systems, lipids and proteins are arranged uniaxially around the mem brane, allowing normal orientation of the molecule backbone relative to the substratum. In combination with isotopic labeling (e.g., 2H, 13C, 15N) this NMR ap proach has successfully been used to determine the complete secondary structure of the M2 channel pep tide and fd coat protein (3), while the orientation of the antibiotic peptide magainin has been resolved in bilayers (5). The complete secondary structure of gram icidin and its dynamic properties in membranes have also been obtained using 2H and 15N NMR (6-9). In addition, the complete structure and orientation of deu terated retinal in bacteriorhodopsin at different states of its photocycle has been resolved (10), The average molecular and orientational order of lipids and peptides in liquid crystalline membranes could also be deter mined unambiguously in this way (11-14).Unfortunately, the generation of structural and ori entational information about membrane proteins has been limited mainly to peptides, since no general methods have been available for aligning native and reconstituted membranes containing larger integral membrane proteins in sufficient quality and quantity without affect...

Dynamics and orientation of N + (CD 3 ) 3 -bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

“…Changing the tilt angle of the membrane to 90°results in a shift in the resonance to Ϫ15 ppm (Fig. 4B) as expected for macroscopically aligned lamellar membranes (30). The observed chemical shift anisotropy is typical for a mixed phosphatidylcholine͞phosphati-dylethanolamine lipid system in its L ␣ phase (30).…”

“…4B) as expected for macroscopically aligned lamellar membranes (30). The observed chemical shift anisotropy is typical for a mixed phosphatidylcholine͞phosphati-dylethanolamine lipid system in its L ␣ phase (30). The broad nature of the 0°and the 90°spectrum can be attributed to the distribution of different lipid types (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) within the sample, each of which exhibits a slightly different chemical shift anisotropy (42).…”

“…nAcChoR membranes were oriented by using an isopotential spinning technique (30). Alignment was achieved through pelletting 1 ml of nAcChoR membranes (1 mg⅐ml -1 ) onto a Mellanex sheet (Agar Scientific, Stansted, U.K.) by centrifigation (90,000 g for 15 h at 4°C) without drying.…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…The ISD method also has proved useful for a variety of biophysical measurements besides polarized FTIR including CD [119,202], ESR [75,76,97], solid-state NMR [104] and X-ray scattering measurements of oriented biomembrane films [106]. In collaboration with Sol Gruner, who was then at Princeton University, ISD prepared films of native photoreceptor membrane were studied as a function of hydration using X-ray diffraction [106].…”

An introduction to MAS NMR spectroscopy on oriented membrane proteins

“…In principle, good spectral resolution can be obtained by using the anisotropic, two-dimensional character of membranes, to prepare macroscopically ordered samples with well oriented lipids and proteins, either by aligning them as bilayers on glass disks (5,7) or by using bicelles (8)(9)(10). Since the signal frequency observed from a second rank tensor interaction (such anisotropic chemical shift, quadrupolar, and dipolar coupling) depends on its orientation with respect to the field of interaction, sharp resonance lines would be observed if all tensors would have the same orientation with respect to the magnetic field B 0 .…”