Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

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.

Hydrogen Bonding Compensation on the Convex Solvent-Exposed Helical Face of IA3, an Intrinsically Disordered Protein.

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 N-terminal domain (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 intrinsically disordered proteins (IDPs) with varied biotechnological applications.

Enzymatic glycoengineering-based spin labelling of cell surface sialoglycans to enable their analysis by electron paramagnetic resonance (EPR) spectroscopy.

A novel method for spin labelling of sialoglycans on the cell surface is described. C9-Azido sialic acid was linked to glycans on live cells via CSTII-catalysed α2,3-sialylation utilizing azido-sialic acid nucleotide as a sialyl donor, which was followed by attachment of a spin label to the azide via click reaction. It enables the study of cell surface sialoglycans by EPR spectroscopy.

Conformational landscape of non-B variants of HIV-1 protease: A pulsed EPR study.

HIV infection is a global health epidemic with current FDA-approved HIV-1 Protease inhibitors (PIs) designed against subtype B protease, yet they are used in HIV treatment world-wide regardless of patient HIV classification. In this study, double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy was utilized to gain insights in how natural polymorphisms in several African and Brazilian protease (PR) variants affect the conformational landscape both in the absence and presence of inhibitors. Findings show that Subtypes F and H HIV-1 PR adopt a primarily closed conformation in the unbound state with two secondary mutations, D60E and I62V, postulated to be responsible for the increased probability for closed conformation. In contrast, subtype D, CRF_AG, and CRF_BF HIV-1 PR adopt a primarily semi-open conformation, as observed for PI-naïve-subtype B when unbound by substrate or inhibitor. The impact that inhibitor binding has on shifting the conformational land scape of these variants is also characterized, where analysis provides classification of inhibitor induced shifts away from the semi-open state into weak, moderate and strong effects. The findings are compared to those for prior studies of inhibitor induced conformational shifts in PI-naïve Subtype B, C and CRF_AE.

Ion-dependent mobility effects of the Fusobacterium nucleatum glycine riboswitch aptamer II via site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR).

Site-directed spin-labeling (SDSL) with continuous wave electron paramagnetic resonance (cw-EPR) spectroscopy was utilized to probe site-specific changes in backbone dynamics that accompany folding of the isolated 84 nucleotide aptamer II domain of the Fusobacterium nucleatum (FN) glycine riboswitch. Spin-labels were incorporated using splinted ligation strategies. Results show differential dynamics for spin-labels incorporated into the backbone at a base-paired and loop region. Additionally, the addition of a biologically relevant concentration of 5 mM  Mg2+, to an RNA solution with 100 mM K+, folds and compacts the structure, inferred by a reduction in spin-label mobility. Furthermore, when controlling for ionic strength, Mg2+ added to the RNA induces more folding/less flexibility at the two sites than RNA with K+ alone. Addition of glycine does not alter the dynamics of this singlet aptamer II, indicating that the full length riboswitch construct may be needed for glycine binding and induced conformational changes. This work adds to our growing understanding of how splinted-ligation SDSL can be utilized to interrogate differential dynamics in large dynamic RNAs, providing insights into how RNA folding and structure is differentially stabilized by monovalent versus divalent cations.

Effects of Hinge-region Natural Polymorphisms on Human Immunodeficiency Virus-Type 1 Protease Structure, Dynamics, and Drug Pressure Evolution.

Multidrug resistance to current Food and Drug Administration-approved HIV-1 protease (PR) inhibitors drives the need to understand the fundamental mechanisms of how drug pressure-selected mutations, which are oftentimes natural polymorphisms, elicit their effect on enzyme function and resistance. Here, the impacts of the hinge-region natural polymorphism at residue 35, glutamate to aspartate (E35D), alone and in conjunction with residue 57, arginine to lysine (R57K), are characterized with the goal of understanding how altered salt bridge interactions between the hinge and flap regions are associated with changes in structure, motional dynamics, conformational sampling, kinetic parameters, and inhibitor affinity. The combined results reveal that the single E35D substitution leads to diminished salt bridge interactions between residues 35 and 57 and gives rise to the stabilization of open-like conformational states with overall increased backbone dynamics. In HIV-1 PR constructs where sites 35 and 57 are both mutated (e.g. E35D and R57K), x-ray structures reveal an altered network of interactions that replace the salt bridge thus stabilizing the structural integrity between the flap and hinge regions. Despite the altered conformational sampling and dynamics when the salt bridge is disrupted, enzyme kinetic parameters and inhibition constants are similar to those obtained for subtype B PR. Results demonstrate that these hinge-region natural polymorphisms, which may arise as drug pressure secondary mutations, alter protein dynamics and the conformational landscape, which are important thermodynamic parameters to consider for development of inhibitors that target for non-subtype B PR.

Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the Kink-Turn Motif in the Glycine Riboswitch by Site-Directed Spin-Labeling.

Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a means for a solution state description of site-specific dynamics and flexibility of large RNAs, facilitating our understanding of the effects of environmental conditions such as ligands and ions on RNA structure and dynamics. Here, the utility and capability of EPR line shape analysis and distance measurements to monitor and describe site-specific changes in the conformational dynamics of internal loop nucleobases as well as helix-helix interactions of the kink-turn motif in the Vibrio cholerae (VC) glycine riboswitch that occur upon sequential K(+)-, Mg(2+)-, and glycine-induced folding were explored. Spin-labels were incorporated into the 232-nucleotide sequence via splinted ligation strategies. Thiouridine nucleobase labeling within the internal loop reveals unambiguous differential dynamics for two successive sites labeled, with varied rates of motion reflective of base flipping and base stacking. EPR-based distance measurements for nitroxide spin-labels incorporated within the RNA backbone in the helical regions of the kink-turn motif are reflective of helical formation and tertiary interaction induced by ion stabilization. In both instances, results indicate that the structural formation of the kink-turn motif in the VC glycine riboswitch can be stabilized by 100 mM K(+) where the conformational flexibility of the kink-turn motif is not further tightened by subsequent addition of divalent ions. Although glycine binding is likely to induce structural and dynamic changes in other regions, SDSL indicates no impact of glycine binding on the local dynamics or structure of the kink-turn motif as investigated here. Overall, these results demonstrate the ability of SDSL to interrogate site-specific base dynamics and packing of helices in large RNAs and demonstrate ion-induced stability of the kink-turn fold of the VC riboswitch.

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts.

The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function of inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed "curled/tucked", "closed", "semi-open" and "wide-open" conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.

The role of select subtype polymorphisms on HIV-1 protease conformational sampling and dynamics.

HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR (15)N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors.

Residue specific partitioning of KL4 into phospholipid bilayers.

KL4, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL4 and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL4 on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL4, and the partitioning depths of specific KL4 residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL4 induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL4 (V.C. Antharam et al., 2009), we are able to distinguish how KL4 affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL4 also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL4 are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.