An EPR "spectroscopic ruler" was developed using a series of a-helical polypeptides, each modified with two nitroxide spin labels. The EPR line broadening due to electron-electron dipolar interactions in the frozen state was determined using the Fourier deconvolution method. These dipolar spectra were then used to estimate the distances between the two nitroxides separated by 8-25 A. Results agreed well with a simple a-helical model. The standard deviation from the model system was 0.9 A in the range of [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] A. This technique is applicable to complex systems such as membrane receptors and channels, which are difficult to access with high-resolution NMR or x-ray crystallography, and is expected to be particularly useful for systems for which optical methods are hampered by the presence of lightinterfering membranes or chromophores.Many studies in structural biology are dependent on the physical techniques to measure distances in proteins and nucleic acids. X-ray crystallography and high-resolution NMR have been useful in determining the three-dimensional structures of relatively simple biological macromolecules. For complex systems such as membrane proteins, fluorescence energy transfer (FET) has been the main alternative for measuring distances up to 80 A. FET has been successful for studies of intermolecular organization in biological systems (1, 2), ligandreceptor interactions (3), and structures of nucleic acids (4).Recently, site-directed spin labeling EPR has become useful for studying proteins (5, 6). One or two native residues are mutated to cysteines, which are then labeled with thiol-specific nitroxide spin labels. This technique can also be used to study local secondary structure (7,8). Nucleic acids also appear to be amenable to spin labeling (9). Although spin labeling has been used to estimate distances in the past (10-12), no EPR "spectroscopic ruler" similar to that developed by Stryer and Haugland (13) for FET has been constructed or tested on model systems.In this work a convenient and accurate EPR method to determine distances between two site-specifically placed nitroxides in the range of 8-25 A in biomacromolecules is presented. In this method the pure dipolar spectrum for two interacting spins in the frozen state is directly Fourier deconvoluted from the dipolar broadened continuous-wave EPR spectrum. The average interspin distance and the variance of its distribution are obtained from this dipolar spectrum.The method was tested using a-helical peptides as a model system. The peptides were alanine-based helices with spinlabeled cysteines substituted for alanines at two locations from 1 to 13 residues apart. There is excellent agreement between the spin-spin distances from a simple model and experimental results in the range of 8-25 A. It is also shown that this method is useful for systems that have impurities of singly labeled species. Although this methodology is complementary to FET, EPR has the advantages of easier...

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the ''basal'' transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in Drosophila. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans, Drosophila, Caenorhabditis elegans, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2's primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on Drosophila chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.

The envelope glycoprotein gp41 from human immunodeficiency virus type 1 (HIV-1) is involved in membrane fusion and virus entry. It contains a functionally important leucine zipper-like heptad repeat region (residues 553-590). To investigate the solution structure and membrane-binding properties of this region, cysteine-substituted variants of a 38-residue peptide derived from the heptad repeat were synthesized and modified with nitroxide spin labels. Analytical equilibrium ultracentrifugation studies indicated it is primarily tetrameric in solution, in contrast to the protein gp160 which is a mixture of trimers and tetramers. Electron paramagnetic resonance (EPR) measurements indicated that the peptide was bound to vesicles containing 10 mol % negatively charged lipids. The peptides were bound parallel to the membrane surface, near the water-membrane interface, in a structure different from the solution structure, most likely as monomers. When Asp, Pro, or Ser was substituted for Ile at the core "a" position of the heptad repeat in the middle of the peptide, the coiled coil was destabilized. In addition, these peptides showed reduced membrane-binding affinities. Thus, mutations that destabilized coiled-coil formation also decreased membrane-binding propensity. These experimental results, taken with previous evidence, suggest two functions for the heptad repeat of gp41 after CD4 binding: (1) to form an extended coiled coil; (2) to provide a hydrophobic face that binds to the host-cell membrane, bringing the viral and cellular membranes closer and facilitating fusion.