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The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.

The magnetization, M(H< or =30 T,0.7< or =T< or =300 K), of (C5H12N)2CuBr4 has been used to identify this system as an S = 1/2 Heisenberg two-leg ladder in the strong-coupling limit, J( perpendicular) = 13.3 K and J( parallel) = 3.8 K, with H(c1) = 6.6 T and H(c2) = 14.6 T. An inflection point in M(H,T = 0.7 K) at half saturation, M(s)/2, is described by an effective XXZ chain. The data exhibit universal scaling behavior in the vicinity of H(c1) and H(c2), indicating that the system is near a quantum critical point.

The flap conformations of two drug resistant HIV-1 protease constructs were characterized by molecular dynamic (MD) simulations and distance measurements with pulsed electron paramagnetic resonance (EPR) spectroscopy. MD simulations accurately regenerate the experimentally determined distance profiles and provide structural interpretations of the EPR data. The combined analyses show that the average conformation of the flaps, the range of flap opening and closing and the flexibility of the flaps differ markedly in HIV-1PR as multiple mutations arise in response to antiviral therapy, providing structural insights into the mechanism of inhibitor resistance.Human Immunodeficiency Virus Type 1 protease (HIV-1PR) is an enzyme responsible for gag-pol processing, an essential step in viral maturation and the lifecycle of HIV-1. Inhibition of the activity of HIV-1PR results in immature virus particles that are non-infectious. 1 As such, this protein represents a major target of AIDS antiviral therapy. Emerging resistance to antiviral inhibitor cocktails, due to high viral mutation rates, represents a significant challenge in AIDS treatment. 2 Analysis of data from the Stanford Drug Resistance Database 3 shows that while polymorphisms in the sequence of HIV-1PR naturally occur, there are regions in the protein sequence that appear invariant under normal evolutionary pressures. These invariant regions coincide with the structural elements of the dimer interface, the active site floor, the P3-P3′ substrate binding region and the β-hairpin loops (AKA, flaps). Strikingly, upon exposure to protease inhibitor (PI) cocktail treatment, numerous mutations develop, with high occurrences near residues 40-56 and 80-90, which correspond to the hairpin flaps and the P3-P3′ substrate binding cleft; respectively (data shown in Supp Info). Amino acid substitutions arise in these regions of the protein from random mutations that alter the ability of a given inhibitor to bind as tightly to the active site pocket, allowing for effective protease function with subsequent viral maturation and proliferation of the mutation. Many of these mutations also alter the kinetics of the protease for the multiple polypeptide cleavage sequences in the gag-pol polypeptide. 4-7It can be readily understood how mutations within the active site pocket reduce inhibitor effectiveness considering that many of the current PIs have been specifically designed to bind tightly to the shape of the active site cavity. However, the mechanism by which mutations that are NOT within the active site cavity modulate PI efficiency remains uncertain. It has been hypothesized that mutations in the elbow and flap regions (residues 36 to 58) may alter either the conformation of the flaps defined as the degree of flap opening or closing, or the mobility of the flaps, or both. [4][5][6] Correspondence to: Carlos Simmerling, carlos.simmerling@stonybrook.edu; Gail E. Fanucci, fanucci@chem.ufl.edu. We have previously shown that site-directed spin labeling (SDSL) and pulsed dou...

Human immunodeficiency virus type 1 (HIV-1) protease plays a fundamental role in the maturation and life cycle of the retrovirus HIV-1, as it functions in regulating post-translational processing of the viral polyproteins gag and gag-pol; thus, is a key target of AIDS antiviral therapy. Accessibility fanucci@chem.ufl.edu. Supporting Information Available: Further experimental details, protein sequences, sample preparation, data and error analyses. This material is available free of charge via the Internet at http://pubs.acs.org. of substrate to the active site is mediated by two flaps, which must undergo a large conformational change from an open to a closed conformation during substrate binding and catalysis. The electron paramagnetic resonance (EPR) method of site-directed spin labeling (SDSL) with double electronelectron resonance (DEER) spectroscopy was utilized to monitor the conformations of the flaps in apo HIV-1 protease (HIV-1PR), subtypes B, C, and F, CRF01_A/E, and patient isolates V6 and MDR 769. The distance distribution profiles obtained from analysis of the dipolar modulated echo curves were reconstructed to yield a set of Gaussian-shaped populations, which provide an analysis of the flap conformations sampled. The relative percentages of each conformer population described as "tucked/curled", "closed", "semi-open", and "wide-open" were determined and compared for various constructs. The results and analyses show that sequence variations among subtypes, CRFs and patient isolates of apo HIV-1PR alter the average flap conformation in a way that can be understood as inducing shifts in the relative populations, or conformational sampling, of the previously described four conformations for HIV-1PR. NIH Public AccessHuman immunodeficiency virus type 1 protease (HIV-1PR), a 99 amino acid homodimeric aspartic protease, plays a fundamental role in the maturation and life cycle of the retrovirus HIV-1, as it functions in regulating post-translational processing of viral polyproteins gag and gag-pol. Consequently, this enzyme is a target of AIDS antiviral therapy given that its inhibition prevents viral maturation. 1 Accessibility of substrate to the active site is mediated by two β-hairpins (a.k.a. the flaps), which undergo a conformational change during entry and catalysis. HIV-1 is categorized into different groups, subtypes, and circulating recombinant forms (CRFs), wherein groups refer to distinctive viral lineages, subtypes to taxonomic groups within a particular lineage, and CRFs to recombinant forms of the virus. 2 Each subtype exhibits a unique set of naturally occurring polymorphisms. Protease inhibitors used in treatment of HIV-1 are often designed with respect to subtype B;3 thus, it is of great importance to understand how subtype polymorphisms alter protein structure, flexibility, and inhibitor efficacy.4 -10Site-directed spin labeling (SDSL) double-electron-electron resonance (DEER), a pulsed electron paramagnetic resonance (EPR) spectroscopy technique, provides a means to monitor the ...

Double electron-electron resonance (DEER), a pulsed electron paramagnetic resonance (EPR) spectroscopy technique, was utilized to characterize conformational population shifts in HIV-1 protease (HIV-1PR) upon interaction with various inhibitors. Distances between spin-labeled sites in the flap region of HIV-1PR were determined, and detailed analyses provide population percentages for the ensemble flap conformations upon interaction with inhibitor or substrate. Comparisons are made between the percentage of the closed conformer seen with DEER and enzymatic inhibition constants, thermodynamic dissociation constants, and the number of hydrogen bonds identified in crystallographic complexes.

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The structure and dynamics of the N-terminal and core regions of BtuB, an outer membrane vitamin B(12) transporter from Escherichia coli, were investigated by site-directed spin labeling. Cysteine mutants were generated by site-directed mutagenesis to place spin labels in the N-terminal region (residues 1-17), the core region (residues 25-30), and double labels into the Ton box (residues 6-12). BtuB mutants were expressed, spin labeled, purified, and reconstituted into phosphatidylcholine. In the presence of substrate (vitamin B(12)), EPR spectroscopy demonstrates that there is a conformational change in the Ton box similar to that seen previously for BtuB in intact outer membranes. The Ton box is positioned within the beta-barrel of BtuB in the absence of substrate (docked configuration) but becomes unfolded and increases its aqueous exposure upon substrate binding (undocked configuration). This conformational change and the similarity in the EPR spectra between reconstituted and native membranes indicate that BtuB is correctly folded and functional in the reconstituted system. The protein segment on the N-terminal side of the Ton box is highly mobile, and it becomes more mobile in the presence of substrate. Side chains in the region C-terminal to the Ton box also show increases in mobility with substrate addition, but position 16 appears to define a hinge point for this conformation change. EPR line shapes and relaxation data indicate that residues 25-30 form a beta-strand structure, which is analogous to the first beta-strand in the cores of the homologous iron transporters. When substrate binds to BtuB, this first beta-strand remains folded. The EPR spectra of double-nitroxide labels within the Ton box are broadened because of dipolar and collisional exchange interactions. The broadening pattern indicates that the Ton box is not helical but is in an extended or beta-strand structure.