Hydrophobic Clusters Regulate Surface Hydration Dynamics of Lipase A.

The surface hydration diffusivity of Lipase A (BSLA) has been characterized by low-field Overhauser dynamic nuclear polarization (ODNP) relaxometry using a series of spin-labeled constructs. Sites for spin-label incorporation were previously designed via an atomistic computational approach that screened for surface exposure, reflective of the surface hydration comparable to other proteins studied by this method, as well as minimal impact on protein function, dynamics, and structure of BSLA by excluding any surface site that participated in greater than 30% occupancy of a hydrogen bonding network within BSLA. Experimental ODNP relaxometry coupling factor results verify the overall surface hydration behavior for these BSLA spin-labeled sites similar to other globular proteins.

Designing surface exposed sites on Bacillus subtilis lipase A for spin-labeling and hydration studies.

Spin-labeling with electron paramagnetic resonance spectroscopy (EPR) is a facile method for interrogating macromolecular flexibility, conformational changes, accessibility, and hydration. Within we present a computationally based approach for the rational selection of reporter sites in Bacillus subtilis lipase A (BSLA) for substitution to cysteine residues with subsequent modification with a spin-label that are expected to not significantly perturb the wild-type structure, dynamics, or enzymatic function. Experimental circular dichroism spectroscopy, Michaelis-Menten kinetic parameters and EPR spectroscopy data validate the success of this approach to computationally select reporter sites for future magnetic resonance investigations of hydration and hydration changes induced by polymer conjugation, tethering, immobilization, or amino acid substitution in BSLA.

Charge Distribution Patterns of IA Impact Conformational Expansion and Hydration Diffusivity of the Disordered Ensemble.

IA is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically disordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based on the intrinsically disordered protein (IDP) parameters of fractional net charge (), net charge density per residue (), and charge patterning (κ), the two domains of IA are defined to occupy different domains within conformationally based subclasses of IDPs, thus making IA 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.

Hydrogen Bonding Compensation on the Convex Solvent-Exposed Helical Face of IA, an Intrinsically Disordered Protein.

IA 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 N-terminal domain (NTD) of IA-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.

Different Biophysical Properties of Cell Surface α2,3- and α2,6-Sialoglycans Revealed by Electron Paramagnetic Resonance Spectroscopic Studies.

Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of azide-modified sialic acid (Neu5Ac9N) and then click reaction-based attachment of a nitroxide spin radical. α2,6-Sialyltransferase (ST) Pd2,6ST and α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N, 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.