Spin diffusion methods in protein structure determination by solid-state NMR

Abstract

<p>Different spin diffusion methods are applied to study protein structure in lipid membranes using solid-state NMR. Proton driven 13C spin diffusion provided unambiguous evidence for the structural rearrangement of colicin la channel domain upon binding to the membrane. Many short distances that exist in the soluble colicin became too long to be detectable in the 2D 13C correlation spectra. The degree of the reduction of the observable short distances is found to depend on the fraction of inter-helical contacts among all short distances. This indicates that these inter-helical distances become longer in the membrane-bound colicin making the corresponding cross peaks undetectable in the membrane-bound colicin spectra. Thus an open topology is formed upon membrane binding of colicin la and a structural model is proposed to represent this process. 1H spin diffusion is a novel method that gives a sensitivity enhancement to the two orders of magnitude compared to the original 2D experiment. This method removes the 13C dimension and detects the sensitive 1H nucleus. The same topology information as the original 2D experiment can be obtained that membrane bound colicin la channel domain has large fraction at the surface of the membrane while having a small fraction inserting into the lipid bilayer. This method is more reliable at the higher labeling level and is generally applicable to the study of membrane protein topology. 1H-driven 13C and 19F spin diffusion are analyzed to provide distance information in addition to spin counting. In the simulations, the overlap integral, F(0), and intermolecular distances between the spins with different orientations are both unknown. By analyzing model compounds with known distances, we determined universal F(0) values for 13C spin diffusion at 5 kHz MAS and 19F spin diffusion at 8 kHz MAS. Moreover, the F(0) value is shown to be approximately inversely proportional to the spinning speed for 13C spin diffusion. With the F(0) value determined, distances can be extracted for structurally unknown peptides. Two membrane peptides, A30F-TMP-M2 in DMPC and PG-1 in POPC, are studied with 19F spin diffusion and the shortest 19F-19F distances are determined from the buildup curve simulation. This method can be applicable to other membrane proteins to measure the intermolecular distances.</p>