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The calculations of Cu(super +) binding energies of ethyl and vinyl amines, phosphines, and arsines using the B3LYP density functional theory (DFT) are presented. Notable differences between nitrogen-, phosphorus-, and arsenic-containing compounds are observed.

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

The effects of solutes on spin-label mobility and protein conformation have been investigated with X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy for spin labels attached to an aqueous-exposed site in the beta-hairpin flap region of HIV-1 protease. Specifically, we examined the effects of glycerol, sucrose, PEG3000, and Ficoll400 for four commonly used nitroxide spin labels and found that the largest perturbations to the EPR line shapes occur for solutions containing PEG3000 and glycerol. From comparisons of the spectral line shapes and distance distribution profiles of spin-labeled HIV-1 protease with and without inhibitor, it was concluded that solutes such as glycerol and PEG3000 alter the line shapes of the spin label in the beta-hairpin flaps of HIV-1 PR by modulation of spin-label mobility through changes in preferential interactions with the solutes. It is noteworthy that the high osmolality of the 40% glycerol solution did not alter the conformation of the flaps as determined from pulsed EPR distance measurements.

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.