Nuclear magnetic resonance spectroscopy in analysis of granulin three-dimensional structure and cysteine bridging

Abstract

Granulin (GRN) structural motif represents a ladderlike stack of β-hairpins reinforced with six parallel disulfide bridges. When GRNs are produced in a recombinant protein expression host (e.g., in bacteria) or via chemical synthesis, the formation of disulfide bridges from thiols undergoing uncontrolled oxidation may be random. As a consequence, the resulting protein could be a mixture of a large number of disulfide species. Incorrectly folded GRNs may behave abnormally in bioassays; therefore isolation and identification of properly structured, chemically homogenous GRN peptides is very important for biological relevance of the GRN effects observed in the tests. Protein nuclear magnetic resonance (NMR) spectroscopy is an excellent tool for identification and characterization of well-structured GRN disulfide species produced in an Escherichia coli expression system. At first, GRN disulfide species are crudely separated by reversed-phase HPLC chromatography. Obtained fractions are screened by 1D (one-dimensional) proton NMR for the presence of well-folded GRN species. The well-folded GRNs are 15 N-labeled and purified, and NMR is used to determine their three-dimensional structure and assign disulfide pairing patterns. Additionally, NMR characterization of model peptides derived from the GRN amino acid sequences can help resolve ambiguities in disulfide bond assignment. This approach was first successfully used to obtain biologically active human GRNs, but it can be easily expanded to GRN peptides from other species and/or generated by other methods.