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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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in diameter enable two-dimensional, solid-state nuclear magnetic resonance studies of lipid-induced conformational changes of uniformly (15)N-labeled Pf1 coat protein in native-like bilayers. The Pf1 helix tilt angles in bilayers composed of two different lipids are not entirely governed by the membrane thickness but could be rationalized by hydrophobic interactions of lysines at the bilayer interface. The anodic aluminum oxide alignment method is applicable to a broader repertoire of lipids versus bicelle bilayer mimetics currently employed in solid-state nuclear magnetic resonance of oriented samples, thus allowing for elucidation of the role played by lipids in shaping membrane proteins., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the "shotgun" approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the (15)N chemical shift and (15)N-(1)H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an (15)N-(15)N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled "seed" spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B (1) fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.

Thermodynamic limit of magnetization corresponding to the intact proton bath usually cannot be transferred in a single cross-polarization contact. This is mainly due to the finite ratio between the number densities of the high- and low-gamma nuclei, quantum-mechanical bounds on spin dynamics, and Hartmann-Hahn mismatches due to rf field inhomogeneity. Moreover, for fully hydrated membrane proteins refolded in magnetically oriented bicelles, short spin-lock relaxation times (T1ρ) and rf heating can further decrease cross polarization efficiency. Here we show that multiple equilibrations-re-equilibrations of the high- and low-spin reservoirs during the preparation period yield an over twofold gain in the magnetization transfer as compared to a single-contact cross polarization (CP), and up to 45% enhancement as compared to the mismatch-optimized CP-MOIST scheme for bicelle-reconstituted membrane proteins. This enhancement is achieved by employing the differences between the spin-lattice relaxation times for the high- and low-gamma spins. The new technique is applicable to systems with short T1ρ's, and speeds up acquisition of the multidimensional solid-state NMR spectra of oriented membrane proteins for their subsequent structural and dynamic studies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To improve the oral bioavailability of patchoulic alcohol in rats by using self-microemulsifying drug delivery systems (SMEDDS)., Method: Patchoulic alcohol was separated and purified from patchoulic oil, and the SMEDDSs including patchoulic alcohol or patchoulic oil were optimized by pseudo-ternary phase diagrams via central composite design-response surface methodology. Pharmacokinetics of both SMEDDSs and patchoulic alcohol itself in rats were investigated., Result: The patchoulic alcohol SMEDDS (Cremophor EL-Tween 80-PEG 400-isopropyl myristate-patchoulic alcohol, 2:2:0.8:1.95:0.65) was considered as the optimized formulation. The mean drop size of the system was 30. 1 nm after diluted 100 folds in water. The average self-microemulsifying time was 142 s. Patchoulic alcohol SMEDDS and patchoulic oil SMEDDS showed no signficant difference in Tmax compared with patchoulic alcohol with around 60 minutes, while the AUCs of both SMEDDSs (2001 745.6 +/- 329 663.6) and (1594 005.6 +/- 280 150.3) microg x min x L(-1) were significantly higher than that of patchoulic alcohol (1 163 153.3 +/- 232 324.3) microg x min x L(-1)., Conclusion: SMEDDS can effectively improve the oral bioavailability of patchoulic alcohol in rats.