CONSTRUCTION OF LOW TEMPERATURE SOLID STATE NMR PROBE

University Of Pennsylvania, Philadelphia PA

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Abstract

The development of a new method for protein structure determination by solid-state NMR is the focus of this research. The methods being developed rely on several features of the protein system. The system should be immobilized on a time scale appropriate to preserve the orientation dependent spin interactions and a uni-axis of orientation of the protein aligned parallel to the applied magnetic field must be present. These criteria are met for proteins bound to lipid bilayers oriented mechanically on glass plates. We are currently evaluating other systems that orient spontaneously in magnetic fields. These systems are composed of mixtures of phospholipids and detergents that form bilayer discs that orient with the long axis perpendicular to the applied magnetic field. These discs are referred to as bicelles. The discs were found to flip when certain paramagnetic ions were added to the bicelles. This orients the discs in the favorable position with the long axis parallel to the applied field. When proteins are inserted into these bicelles, measurements pertaining to the protein backbone structure can be made from the 15N and 1H orientations at the amide sites. Several membrane proteins have been successfully incorporated into the bicelle system. These include the coat protein from the filamentous bacteriophage fd, the mercury transport protein merT, the HIV-1 accessory protein Vpu and the M2 peptide from the acetycholine receptor. Characterization of these proteins is proceeding using the bicelle technique for solid-state NMR structure determination. Other methods of orienting the bicelle discs are being pursued. The use of an EF hand, calcium binding protein motif for the purpose of binding the paramagnetic metal is being tested. This system will direct the paramagnetic metal to the designated site of binding, orient the bicelle with the appropriate axis and leave the protein to be studied free of any interaction with the paramagnetic ion.