Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.Keywords: site-directed spin labeling; EPR spectroscopy; side-chain conformation Supplemental material: see www.proteinscience.org Site-directed spin labeling (SDSL) has become a widely used tool to study protein structure, dynamics, and interactions (for reviews, see Hubbell et al. 1996Hubbell et al. , 1998Hubbell et al. , 2000Columbus and Hubbell 2002;Klug and Feix 2004;Fanucci and Cafiso 2006). The SDSL approach requires a cysteine residue to be introduced at a selected site and subsequently modified with a nitroxide reagent to generate a disulfidelinked nitroxide side chain. The most commonly employed spin-label side chain is designated R1 (Fig. 1). The EPR spectra of R1 directly encode information on the motion of the nitroxide, which in turn reflects structure and dynamics of the local protein environment at the labeling site. A primary goal in developing the SDSL method is to enable interpretation of EPR spectra in terms of protein structure and dynamics using well-characterized proteins as models. Success in this regard will allow SDSL to be used to infer these features in poorly characterized proteins that are challenging to other methods.At the present time, simple one-component EPR spectra that arise from R1 on solvent-exposed surfaces of a-helices and in ordered turns are reasonably well understood (Guo et al. 2007). At these sites, the motion of R1 is determined by the internal motion of the side chain and local backbone dynamics (Columbus et al. 2001), and the EPR spectra provide a means for mapping backbone dynamics (Columbus and Hubbell 2004 Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

The polysulfated polyxylan HOE/BAY946, which has been tested in two pilot studies in ARC/AIDS patients and in asymptomatic HIV carries in Germany, was believed to act by inhibiting virus attachment to the cell. However, the drug was also found to reduce the amount of HIV particles released from infected peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, preincubation of PBMC with the drug led to a partial inhibition of a following HIV infection, suggesting that the drug also affects virus entry. Electron Paramagnetic Resonance (EPR) measurements on uninfected human lymphocytes using 5-proxyl-nonane as spin label demonstrated smaller hyperfine coupling constant (aN) values in the presence of HOE/BAY946 or dextran sulfate 5000. Accordingly, h-1p/h-1H ratios were decreased, indicating increased plasma membrane hydrophobicity and a membrane-stabilizing effect of the drugs. Culture of the chronically HIV-infected monocytic cell line U937/HIV-2D194 in the presence of HOE/BAY946 specifically and drastically reduced the release of virions and the intracellular synthesis of viral proteins as determined by radioimmunoprecipitation and reverse transcriptase assays. In conclusion, although the EPR studies showed a physico-chemical effect on membrane polarity, HOE/BAY946 and dextran sulfate clearly affect processes beyond the cell membrane. Thus, in contrast to previous reports suggesting that polysulfated sugars affect HIV only by inhibiting virus binding to uninfected cells, they clearly inhibit HIV in infected cells as well and appear to have a pleiotropic mode of action. Such drugs may be less likely to result in viral resistance after prolonged application than substances acting only on one step in the life cycle of the virus.

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ESR relaxation measurements were carried out for a dilute toluene solution of in-chain-labeled polystyrene containing double bond in the main chain next to the spin label. The temperature was varied from -20 to +93 °C. It was found that the correlation time is larger by an order of magnitude than that of the unlabeled polystyrene, which can be explained by the influence of double bond on the segmental motion. The number of monomer units involved in the ESR relaxation process was estimated as 2-4. The preparation of spin-labeled polystyrene copolymer was also described.

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