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.

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.

Natural Polymorphisms D60E and I62V Stabilize a Closed Conformation in HIV-1 Protease in the Absence of an Inhibitor or Substrate.

HIV infection remains a global health issue plagued by drug resistance and virological failure. Natural polymorphisms (NPs) contained within several African and Brazilian protease (PR) variants have been shown to induce a conformational landscape of more closed conformations compared to the sequence of subtype B prevalent in North America and Western Europe. Here we demonstrate through experimental pulsed EPR distance measurements and molecular dynamic (MD) simulations that the two common NPs D60E and I62V found within subtypes F and H can induce a closed conformation when introduced into HIV-1PR subtype B.

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.

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.

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.

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 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 click reaction. It enables the study of cell surface sialoglycans by EPR spectroscopy.

A metabolically engineered spin-labeling approach for studying glycans on cells.

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 -acetylglucosamine (GlcNAc) were targeted for spin labelling.

Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance.

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.

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.

Block Copolymer Sequence Inversion through Photoiniferter Polymerization.

Block copolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization are often restricted to a specific comonomer blocking sequence that is dictated by intermediate radical stability and relative radical leaving group abilities. Techniques that provide alternative pathways for reinitiation of thiocarbonylthio-terminated polymers could allow access to block copolymer sequences currently unobtainable through the RAFT process. We report a method for preparing "inverted" block copolymers, whereby the traditional order of monomer addition has been reversed through the use of photoiniferter-mediated radical polymerization.

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.

Probing Membrane Hydration at the Interface of Self-Assembled Peptide Amphiphiles Using Electron Paramagnetic Resonance.

The relative hydrophilicity at the interface of a nanoparticle was measured utilizing electron paramagnetic resonance (EPR) spectroscopy. The supramolecular structure was assembled from spin-labeled peptide amphiphiles (PA) derived from carboxy anhydrides (NCA). Cyanuric chloride, or 2,4,6-trichloro-1,3,5-triazine (TCT), was used as a modular platform to synthesize the spin-labeled, lipid-mimetic macroinitiator used for the ring-opening polymerization of γ-benzyl-l-glutamic acid NCA to produce polyglutamate--dodecanethiol.

Synthetic upcycling of polyacrylates through organocatalyzed post-polymerization modification.

The direct transformation of commercially available commodity polyacrylates into value-added materials was achieved. We demonstrate how 1,5,7-triazabicyclo[4.4.

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.

Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes.

Dynamic nuclear polarization (DNP) enhanced solid-state NMR can provide orders of magnitude in signal enhancement. One of the most important aspects of obtaining efficient DNP enhancements is the optimization of the paramagnetic polarization agents used. To date, the most utilized polarization agents are nitroxide biradicals.

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.

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields.

High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(∼94 GHz) and D-band (∼140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to α-helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA.

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.

Spin labeling and Double Electron-Electron Resonance (DEER) to Deconstruct Conformational Ensembles of HIV Protease.

An understanding of macromolecular conformational equilibrium in biological systems is oftentimes essential to understand function, dysfunction, and disease. For the past few years, our lab has been utilizing site-directed spin labeling (SDSL), coupled with electron paramagnetic resonance (EPR) spectroscopy, to characterize the conformational ensemble and ligand-induced conformational shifts of HIV-1 protease (HIV-1PR). The biomedical importance of characterizing the fractional occupancy of states within the conformational ensemble critically impacts our hypothesis of a conformational selection mechanism of drug-resistance evolution in HIV-1PR.

A method for dynamic nuclear polarization enhancement of membrane proteins.

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4 . Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL.

Site-directed spin-labeling strategies and electron paramagnetic resonance spectroscopy for large riboswitches.

Genetic regulation effected by RNA riboswitches is governed by ligand-induced structural reorganization with modulation of RNA conformation and dynamics. Characterization of the conformational states of riboswitches in the presence or absence of salts and ligands is important for understanding how interconversion of riboswitch RNA folding states influences function. The methodology of site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is suitable for such studies, wherein site-specific incorporation of a nitroxide radical spin probe allows for local dynamics and conformational changes to be investigated.

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.