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The surface hydration diffusivity of Bacillus subtilis 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. Here, by plotting the ODNP parameters of relative diffusive water versus the relative bound water, we introduce an effective "phase-space" analysis, which provides a facile visual comparison of the ODNP parameters of various biomolecular systems studied to date. We find notable differences when comparing BSLA to other systems, as well as when comparing different clusters on the surface of BSLA. Specifically, we find a grouping of sites that correspond to the spin-label surface location within the two main hydrophobic core clusters of the branched aliphatic amino acids isoleucine, leucine, and valine cores observed in the BSLA crystal structure. The results imply that hydrophobic clustering may dictate local surface hydration properties, perhaps through modulation of protein conformations and samplings of the unfolded states, providing insights into how the dynamics of the hydration shell is coupled to protein motion and fluctuations. 

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. Analysis of molecular dynamic simulations of the impact of substitutions on the secondary structure agree well with experimental findings. We propose that this computationally guided approach for choosing spin-labeled EPR reporter sites, which evaluates relative surface accessibility coupled with hydrogen bonding occupancy of amino acids to the catalytic pocket via atomistic simulations, should be readily transferable to other macromolecular systems of interest including selecting sites for paramagnetic relaxation enhancement NMR studies, other spin-labeling EPR studies or any method requiring a tagging method where it is desirable to not alter enzyme stability or activity. 

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. 

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur. 

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. 

A new method is developed for spin labelling of cell surface sialoglycansviaenzymatic engineering to install azido-sialic acid and then click reaction to attach spin. EPR studies of labeled cells revealed the environments around the sialoglycans. 

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells. This approach exploited the incorporation of azides into cell surface glycans followed by a click reaction with a new nitroxide spin label. Both sialic acid and N -acetylglucosamine (GlcNAc) were targeted for spin labelling. Although each of these moieties experiences a diverse and heterogeneous glycan environment, their EPR spectra and hence mobility are both characterized as a linear combination of two distinct spectra where one component reflects a highly mobile or uncrowded micro-environment with the second component reflecting more restricted motion, reflective of increased crowding and packing within the glycocalyx. What differs among the spectra of the targeted glycans is the relative percentage of each component, with sialic acid moieties experiencing on average an ∼80% less crowded environment, where conversely GlcNAc/GalNAz labeled sites reported on average a ∼50% more crowded environment. These distinct environments are consistent with the organization of sugar moieties within cellular glycans where some residues occur close to the cell membrane/protein backbone ( i.e. more restricted) and others are more terminal in the glycan ( i.e. more mobile). Strikingly, different cell lines displayed varied relative populations of these two components, suggesting distinctive glycan packing, organization, and composition of different cells. This work demonstrates the capability of SDSL EPR to be a broadly useful tool for studying glycans on cells, and interpretation of the results provides insights for distinguishing the differences and changes in the local organization and heterogeneity of the cellular glycocalyx. 

Multidrug resistance continues to be a barrier to the effectiveness of highly active antiretroviral therapy in the treatment of human immunodeficiency virus 1 (HIV-1) infection. Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10–20 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron–electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-naïve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter Ki and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.