Structure of high molecular weight protein systems by solution NMR spectroscopy

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Introduction***Multinuclear (1H, 13C, 15N) multidimensional solution NMR spectroscopy is a powerful method for determining protein structure at atomic resolution. Studies of systems >30 kDa are hampered by rapid signal decay, though this can be partially circumvented by deuterating the protein of interest (replacing 1H with 2H). Most solution NMR experiments require 1H atoms, however, so these must be judiciously re-introduced. This is currently achieved by adding appropriately labeled amino acids (or their precursors) to the growth media, which are then incorporated into 1H-13C-labeled methyl groups (in Ala, Ile, Leu, Met, Thr, or Val) in an otherwise deuterated protein. Rapid rotation about the methyl C-C axis greatly attenuates signal decay in methyl groups (almost 10-fold), making them ideal probes for solution NMR. The obvious problem with the approach is the lack of obtainable structural information for the sidechains of non-methyl-containing amino acid residues (Cys, Asp, Glu, Phe, His, Lys, Asn, Pro, Gln, Arg, Ser, Trp, and Tyr).******Rationale***Rapid signal decay in 1H-13C groups precludes solution NMR of big systems except in the case of methyl groups. However, 1H-12C groups are readily observable. Magnetization originating on 1H-12C groups can be transferred to methyl 1H-13C groups or backbone amide 1H-15N groups through the nuclear Overhauser enhancement (NOE), yielding important structural information at all of these sites. We have demonstrated the feasibility of this approach in our preliminary data.******Objectives***1) Use E. coli as an expression system to produce deuterated protein with optimally located 1H-12C groups.***We will find optimal carbon sources for E. coli to strategically incorporate 1H-12C groups into otherwise highly deuterated proteins. A model peptide system will be developed to quantitate isotope incorporation patterns at every atom in every amino acid using NMR. This will allow us to understand how the substrates are processed in the central metabolic and amino acid biosynthetic pathways. We will initially examine pyruvate, rhamnose, and fumarate as carbon sources, along with oxalate and malonate as metabolic inhibitors to limit isotope scrambling.******2) Determine high-resolution structures of challenging proteins by solution NMR spectroscopy.***An optimal isotope labeling strategy will be used to produce the integral membrane protein, PagP. A high-resolution structure will be determined on the basis of NOEs between backbone amide 1H-15N, methyl 1H-13C, and 1H-12C sites introduced in this proposal. This will allow us to characterize the active site of PagP to determine its catalytic mechanism.******Impact***The proposed research is widely applicable, extending the range of big systems (like membrane proteins, multi-domain proteins, and protein complexes) that can be studied by solution NMR and greatly improving the quality of structures that can be obtained.********