Evidence for Helical Structure in a Tetramer of α2-8 Sialic Acid: Unveiling a Structural Antigen

Characteristic H-bonding patterns define secondary structure in proteins and nucleic acids. We show that similar patterns apply for α2-8 sialic acid (SiA) in H₂O and that H-bonds define its structure. A ¹⁵N,¹³C α2-8 SiA tetramer, (SiA)₄, was used as a model system for the polymer. At 263 K, we detected intra-residue through-H-bond <i>J</i> couplings between ¹⁵N and C8 for residues R-I–R‑III of the tetramer, indicating H-bonds between the ¹⁵N’s and the O8’s of these residues. Additional <i>J</i> couplings between the ¹⁵N’s and C2’s of the adjacent residues confirm the putative H-bonds. NH groups showing this long-range correlation also experience slower ¹H/²H exchange. Additionally, detection of couplings between H7 and C2 for R-II and R-III implies that the conformations of the linkers between these residues are different than in the monomers. These structural elements are consistent with two left-handed helical models: 2 residues/turn (2₄ helix) and 4 residues/turn (1₄ helix). To discriminate between models, we resorted to ¹H,¹H NOEs. The 2₄ helical model is in better agreement with the experimental data. We provide direct evidence of H-bonding for (SiA)₄ and show how H-bonds can be a determining factor for shaping its 3D structure

SDS-PAGE and Western blot (right panel) comparison of different α-PI's: 1- protein ladder; 2 and 6 - pd-α-PI standard; 3 - deglycosylated pd-α-PI; 4 - α-PI from (eluted from TALON beads); 5 - r-α-PI in the supernatant from D1526

<p><b>Copyright information:</b></p><p>Taken from "Expression of human α-proteinase inhibitor in "</p><p>http://www.microbialcellfactories.com/content/6/1/34</p><p>Microbial Cell Factories 2007;6():34-34.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2186354.</p><p></p

SDS-PAGE and Western blot (right panel) analysis of α-PI expression in D1526

<p><b>Copyright information:</b></p><p>Taken from "Expression of human α-proteinase inhibitor in "</p><p>http://www.microbialcellfactories.com/content/6/1/34</p><p>Microbial Cell Factories 2007;6():34-34.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2186354.</p><p></p> 1-protein ladder, 2 and 10 - pd-α-PI standard, 3–5 - supernatant from growth of the transformant #1 (30, 20, and 10 μL respectively), 6 – supernatant from growth of PYRG-transformant, 7–9 - supernatant from growth of the transformatant #2 (the 30, 20, and 10 μL respectively)

Diagram of the fusion region between glucoamylase (GLA) and α-PI coding region (A1-PI) within the pAN56-1 expression vector showing the KEX2 cleavage sequence (see abbreviations in the text)

<p><b>Copyright information:</b></p><p>Taken from "Expression of human α-proteinase inhibitor in "</p><p>http://www.microbialcellfactories.com/content/6/1/34</p><p>Microbial Cell Factories 2007;6():34-34.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2186354.</p><p></p