Proton-deuteron amide exchange studies by MS and NMR
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Many proteins of biological interest are large, or difficult to express with uniform magnetically active isotopic labels, making them inaccessible to structural study by conventional Nuclear Magnetic Resonance (NMR) methods. A less conventional approach relies on sparse labeling with isotopes in specific amino acid types, but this approach requires new resonance assignment strategies that don’t rely on the presence of isotopic labels in sequential backbone sites. The goal of this thesis is to develop a new protein assignment strategy applicable to a sparsely labeled sample. The approach combines NMR and Mass Spectrometry (MS) and relies on the ability of both methods to monitor the rates of exchange of an amide proton for a water deuteron. MS can identify the peptide sequence which contains the exchanged amides while NMR can provide resolved amide proton signals which reflect the amount of exchange. By correlating amide exchange rates, from data on the native protein and from data on derived peptides, we achieve assignment of NMR peaks to specific positions in the protein sequence. We selected the glycosyltransferase, ST6Gal1 as a long-term objective. This is a 38 kDa glycosylated protein that is not readily expressed in E. Coli. We have also used a more easily expressed 15 kDa lectin, Galectin-3, as an intermediate target on which to demonstrate our methodology. We have successfully 15demonstrated the utility of our assignment strategy on a N phenylalanine labeled sample of Galectin-3, and have demonstrated an ability to acquire data on ST6Gal1 labeled in specific amino acids. We expect the new methodology to open NMR-based structural investigations for a class of proteins that has been largely inaccessible to structural biology investigation in the past.