Hydrophobic Clusters Regulate Surface Hydration Dynamics of Bacillus subtilis Lipase A.

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 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.

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. Specifically, D60E alters the conformation in subtype B through the formation of a salt bridge with residue K43 contained within the nexus between the flap and hinge region of the HIV-1 PR fold. On the other hand, I62V modulates the packing of the hydrophobic cluster of the cantilever and fulcrum, also resulting in a more closed conformation.

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. 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.

Charge Distribution Patterns of IA3 Impact Conformational Expansion and Hydration Diffusivity of the Disordered Ensemble.

IA3 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 (FNC), net charge density per residue (NCPR), and charge patterning (κ), 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 insights into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution patterns within a conformational subclass can impact bound water hydration dynamics, thus possibly offering an alternative thermodynamic property that can encode conformational binding and behavior of IDPs and liquid-liquid phase separations.

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.

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 (Neu5Ac9N3) 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 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 average 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 the spin-label and sialic acid motion for 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 the 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.

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.

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. 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.

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.

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 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.

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.

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. Specifically, thiocarbonylthio photolysis of xanthate- and dithiocarbamate-functional macromolecular chain transfer agents (macro-CTAs) led to the direct formation of leaving group macroradicals otherwise unaffordable by an addition-fragmentation mechanism. We believe this method could provide a route to synthesize multiblock copolymers of synthetically challenging comonomer sequences.

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 N-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-b-dodecanethiol2. Through static and dynamic light scattering, as well as transmission electron microscopy, PAs with DP of 50 and 17 were shown to assemble into stable nanoparticles with an average hydrodynamic radius of 117 and 84 nm, respectively. Continuous wave EPR spectroscopy revealed that the mobility parameter (h-1/h0) and 2Aiso of the nitroxide radical increased with increasing pH, in concert with the deprotonation of the PE side chains and associated helix-coil transition. These results are consistent with an increase in the relative hydration and polarity at the nanoparticle interface, which would be dependent on the secondary structure of the polypeptide. This research suggests that a pH stimulus could be used to facilitate water diffusion through the membrane.

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[4.4.0]dec-5-ene, serving as a nucleophilic catalyst, can be used to catalyze acyl substitution reactions of acrylic polymers in the presence of alcohol and amine nucleophiles. Furthermore, we found that organocatalytic transesterification exhibits high selectivity towards sterically unhindered esters, thus providing a new route towards site-selective acyl substitution of macromolecular materials. Combining this methodology with reversible-deactivation radical polymerization (RDRP) techniques such as reversible addition-fragmentation chain-transfer (RAFT) polymerization allowed for the precise functionalization of sterically-differentiated acrylic copolymers and polymeric chain ends. We envision this approach to expedite functional polymer synthesis and provide access to functional macromolecules prepared from inexpensive, hydrolytically-stable polymeric precursors.

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.

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. However, the efficiency of these polarization agents is diminished when used with samples other than small molecule model compounds. We recently demonstrated the effectiveness of nitroxide labeled lipids as polarization agents for lipids and a membrane embedded peptide. Here, we systematically characterize, via electron paramagnetic (EPR), the dynamics of and the dipolar couplings between nitroxide labeled lipids under conditions relevant to DNP applications. Complemented by DNP enhanced solid-state NMR measurements at 600 MHz/395 GHz, a molecular rationale for the efficiency of nitroxide labeled lipids as DNP polarization agents is developed. Specifically, optimal DNP enhancements are obtained when the nitroxide moiety is attached to the lipid choline headgroup and local nitroxide concentrations yield an average e(-)-e(-) dipolar coupling of 47 MHz. On the basis of these measurements, we propose a framework for development of DNP polarization agents optimal for membrane protein structure determination.

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. For sample volumes of ∼50 µL, concentration sensitivities of 2-20 µM were achieved, representing a ∼10-fold enhancement compared to a cylindrical TE011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.

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.