Research

Scientists from the IQFR and the UCLM have published the 1st characterization by NMR of the snow flea antifreeze protein which is formed exclusively of polyproline II helices & is crucially stabilized by Cα-H ||| O=C H-bonds.

Proteins are highly useful biochemical catalysts and structural materials. Complex protein structures are built up of three basic building blocks: α-helices, β-hairpins and polyproline II (PPII) helices. Whereas the rules governing the formation of α-helices and β-hairpins are well understood to the point that it is possible to design them de novo, our understanding of PPII helical stability and folding is much less complete. This is important because PPII helices form collagen, the chief protein in ligaments and bones, and since transient PPII helices are believed to be present in intrinsically disordered proteins and may be key for amyloid formation and other pathological and physiological processes.

The snow flea antifreeze protein (sfAFP), which consists of a bundle of six PPII helices, is an attractive model system. Whereas attempts in other laboratories to clone and express this protein in bacteria have hitherto failed, our recent successful production of recombinant sfAFP labeled with 13C & 15N has now enabled its thorough characterization by NMR spectroscopy.

Our results reveal that the PPII helical bundle structure has a unique set of chemical shifts and J-couplings. Despite a high content (46%) of flexible glycine residues (a Gly residue = -HN-CαH2-CO-), the protein is surprisingly rigid and conformationally stable. Distinct magnetic & chemical environments for the two Gly α-hydrogens and density functional theory (DFT) calculations indicate that 28 Gly CαH ||| O=C hydrogen bonds stabilize sfAFP. Moreover, NMR relaxation methods, gel filtration, mass spectrometry and analytical ultracentrifugation show that sfAFP exists as a dimer in solution. Our structural model of this dimer indicates that the burial of hydrophobic groups and the formation additional hydrogen bonds at the dimer interface will provide additional stability. This could also explain how the protein protects its ice-interacting face from undesired aggregations. Taken together, these results provide the bases to understand PPII helical bundle stability and identify these structures in intrinsically disordered proteins and proteins forming liquid microdroplets.

Reference: Treviño MÁ, Pantoja-Uceda D, Menéndez M, Gomez MV, Mompeán M, Laurents DV. " The Singular NMR Fingerprint of a Polyproline II Helical Bundle." J Am Chem Soc. 2018 Nov 29. doi: 10.1021/jacs.8b05261.

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