BIOSYNTH OF BACTERIAL CELL WALL COMPONENTS: STRUCT OF D ALANYL CARRIER PROTEIN

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The incorporation of D-alanine into membrane-associated D-alanyl-lipoteichoic in Lactobacillus casei requires the 56 kDa D-alanine-D-alanyl carrier protein ligase (Dcl) and the 8.9 kDa D-alanyl carrier protein (Dcp). To understand the structure-function relationships of the carrier protein, an analysis of the solution structure of this carrier protein has been undertaken. This structure is required for understanding the acceptor and donor reaction specificities of the carrier protein in the D-alanine incorporation system. All attempts to determine the structure of this carrier protein by crystallographic methods have been unsuccessful. With the acyl carrier protein from E. coli, solution NMR was used to establish the structure of this carrier protein involved in fatty acid biosynthesis. Since the D-alanyl carrier protein (Dcp) is a homolog of this ACP, we have decided to use NMR for the determination of the solution conformation. Homonuclear 2D NMR experiments have provided partial sequential assignment of Dcp. Studies of uniformly labeled 15N Dcp (96% by mass spectrometry) have been undertaken. A 15N homonuclear single quantum coherence (HSQC) experiment obtained at the University of Chicago shows that Dcp is suitable for NMR analysis using 3D double resonance spectroscopy. Excellent water suppression was achieved with the aid of gradients and shape pulse technology at 600 MHz and concentration of 0.5 mM. Expected observable 15N-1H resonances are in good correlation with the amino acid content of th protein. To achieve our goal of 85-95% sequence assignment, we would require a 3D NOESY-HSQC with mixing time optimized for maximum sequential assignment and a 3D TOCSY-HSQC with a maximum isotropic mixing time. These experiments will need to be run using gradients and water flip back techniques for water suppression. Neither of these techniques is available in the biomolecular NMR facility at Northwestern University.