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IA3 is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically dis-ordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based upon the intrinsical-ly disordered proteins (IDPs) parameters of fractional net charge (FNC), of net charge density per residue (NCPR) and of charge patterning (), the two domains of IA3 are defined to occupy different domains within conformationally based subclasses of IDPs; thus, making IA3 a bimodal-domain IDP. Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and low-field Overhauser dynamic nuclear polarization (ODNP) spectroscopy results show that these two domains possess different degrees of compaction and hydration diffusivity behavior. This work suggests that SDSL EPR line shapes – analyzed in terms of their local tumbling volume (VL) – provide insight into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution pat-terns within a conformational subclass can impact bound water hydration dynamics; thus, possibly offering an alter-native thermodynamic property that can encode conforma-tional binding and behavior of IDPs and liquid-liquid phase separations. 

Saccharomyces cerevisiae IA3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2-32 when bound to YPRA, with residues 33-68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the NTD of IA3-YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution. Although nearly all substitutions decreased TFE-induced helicity compared to wild-type (WT), each construct did retain helical character in the presence of 30% (v/v) TFE and retained disorder in the absence of TFE. The NTDs of 8 different Saccharomyces species have nearly identical amino acid sequences, indicating that the NTD of IA3 may be highly evolved to adopt a helical fold when bound to YPRA and in the presence of TFE but remain unstructured in solution. Only one natural amino acid substitution explored within the solvent exposed face of the NTD of IA3 induced TFE-helicity greater than the WT sequence. However, chemical modification of a cysteine by a nitroxide spin label that contains an acetamide side chain did enhance TFE-induced helicity. This finding suggests that non-natural amino acids that can increase hydrogen bonding or alter hydration through side chain interactions may be important to consider when rationally designing IDPs with varied biotechnological applications. 

Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of an azide-modified sialic acid (Neu5Ac9N3) and then click reaction-based attachment of a nitroxide spin radical. An α2,6-sialyltransferase (ST) Pd2,6ST and an α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N3, respectively. The spin-labeled cells were analyzed by X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to gain insights into the dynamics and organizations of cell surface α2,6- and α2,3-sialoglycans. Simulations of the EPR spectra revealed fast- and intermediate-motion components for the spin radicals in both sialoglycans. However, α2,6- and α2,3-sialoglycans in HeLa cells possess different distributions of the two components, e.g., a higher average population of the intermediate-motion component for α2,6-sialoglycans (78%) than that for α2,3-sialoglycans (53%). Thus, the average mobility of spin radicals in α2,3-sialoglycans was higher than that in α2,6-sialoglycans. Given the fact that a spin-labeled sialic acid residue attached to the 6-O-position of galactose/N-acetyl-galactosamine would experience less steric hindrance and show more flexibility than that attached to the 3-O-position, these results may reflect the differences in local crowding/packing that restrict spin-label and sialic acid motion for the α2,6-linked sialoglycans. The studies further suggest that Pd2,6ST and CSTII may have different preferences for glycan substrates in the complex environment of extracellular matrix. The discoveries of this work are biologically important as they are useful for interpreting the different functions of α2,6- and α2,3-sialoglycans and indicate the possibility of using Pd2,6ST and CSTII to target different glycoconjugates on cells.