The crystal structure of the conserved core of HIV-1 Nef has been determined in complex with the SH3 domain of a mutant Fyn tyrosine kinase (a single amino acid substitution, Arg-96 to isoleucine), to which Nef binds tightly. The conserved PxxP sequence motif of Nef, known to be important for optimal viral replication, is part of a polyproline type II helix that engages the SH3 domain in a manner resembling closely the interaction of isolated peptides with SH3 domains. The Nef-SH3 structure also reveals how high affinity and specificity in the SH3 interaction is achieved by the presentation of the PxxP motif within the context of the folded structure of Nef.

A simple and effective device for investigating heat-induced denaturation of proteins by electrospray ionization mass spectrometry is described. Results are presented for the denaturation as a function of temperature and solution pH of bovine ubiquitin and bovine cytochrome c. These results are in concert with and extend the earlier results of LeBlanc et al. (Org. Mass Spectrom. 1991, 26, 831). The cooperative effects of pH and temperature on the denaturation of ubiquitin and cytochrome c were investigated. Electrospray ionization mass spectrometry is also shown to be a useful probe of the reversibility of heat-induced denaturation of proteins. Finally, it is demonstrated that heat-induced denaturation can be used to improve the mass spectrometric response of proteins that do not normally yield useful spectra when the solubilized protein is electrosprayed at ambient temperatures.

A complex of two TFIID TATA box-binding protein-associated factors (TA FIIs) is described at 2.0A resolution. The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix. Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules. In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure.

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Positive ion electrospray ionization mass spectra of polypeptides are usually obtained from solutions that are acidified and therefore contain relatively high concentrations of anions. The present study describes an investigation of the effects of these ubiquitous anions on the positive ion electrospray ionization mass spectra of peptides and proteins. Certain anionic species in the spray solutions were observed to cause a marked decrease in the net average charge of peptide and protein ions in the mass spectra compared to the average charge measured in the absence of these anions. This charge neutralization effect was found to depend solely on the nature of the anionic species and was independent of the source of the anion (acid or salt), with the propensity for neutralization following the order: CCl3COO- > CF3COO- > CH3COO- approximately Cl-. A mechanism for the observed charge reduction effect is proposed that involves two steps. The first step occurs in solution, where an anion pairs with a positively charged basic group on the peptide. The second step occurs during the process of desolvation or in the gas phase, where the ion pair dissociates to yield the neutral acid and the peptide with reduced charge state. The different propensities for charge neutralization of the different anionic species is presumed to reflect the avidity of the anion-peptide interaction. These findings demonstrate that any attempt to correlate the distribution of charge states observed on proteins in the gas phase (by positive ion electrospray ionization mass spectrometry) with the net charge residing on the protein in solution will require that the described anion effect be taken into account. In addition, it appears that some control over the distribution of charge states on peptides and protein ions can be exercised by an appropriate choice of anion in the electrospray solution.

Tissue plasminogen activator binds to endothelial cells via the calcium-regulated phospholipid-binding protein annexin II, an interaction that is inhibited by the prothrombotic amino acid homocysteine. We sought to identify the tissue plasminogen activator binding domain of annexin II and to determine the mechanism of its modulation by homocysteine. Tissue plasminogen activator binding to immobilized annexin II was inhibited by intact fluid phase annexin II but not by its "core" fragment (residues 25-339). Two overlapping "tail" peptides specifically blocked 65-75% of binding. Localization of the tissue plasminogen activator binding domain was confirmed upon specific inhibition by the hexapeptide LCKLSL (residues 7-12). Expressed C9G annexin II protein failed to support tissue plasminogen activator binding, while binding to C133G, C262G, and C335G was equivalent to that of wild type annexin II. Upon exposure to homocysteine, annexin II underwent a 135 ؎ 4-Da increase in mass localizing specifically to Cys 9 and a 60 -66% loss in tissue plasminogen activator-binding capacity (I 50 ‫؍‬ 11 M). Upon treatment of cultured endothelial cells with [35 S]homocysteine, the dithiothreitolsensitive label was recovered by immunoprecipitation with anti-annexin II IgG. These data provide a potential mechanism for the prothrombotic effect of homocysteine by demonstrating direct blockade of the tissue plasminogen activator binding domain of annexin II.

We have identified the site of molecular interaction between nitric oxide (NO) and p21 ras responsible for initiation of signal transduction. We found that p21 It is well known that signal transduction pathways initiated by extracellular ligands are dependent on protein-protein interactions for propagation and amplification of their signal. Many of these interactions lead to phosphorylation events. For example, receptor-tyrosine kinases require a series of protein interactions utilizing SH2 and SH3 domains of adaptor proteins to generate an activated form of p21 ras , a critical signaling enzyme (1-3).Recent studies have identified reactive free radicals as central participants in certain signaling events (4 -7). Enhancing free radical destruction, either enzymatically or chemically, prevented ligand-stimulated transcription factor (8) and mitogen-activated protein (MAP) 1 kinase (4) activation and also prevented smooth muscle cell mitogenesis and chemotaxis (4). Thus, a role is emerging for reactive free radicals in mediating signal transduction.Among the many recently discovered functions of NO, a role in signaling has surfaced (9). Although soluble guanylyl cyclase is an important target of NO in mediating some of its physiologic functions such as the regulation of blood pressure (10, 11), other signaling events, some culminating in transcriptional activation, may be cGMP-independent (12-15). Our studies have focused on how NO initiates cGMP-independent signaling within cells (16,17). We have identified p21ras as a critical target of NO and other redox modulators (17-19). Here, we sought an understanding of the structural basis of the NOp21 ras interaction in the hope of gaining insight into how redox signaling is achieved. MATERIALS AND METHODS Preparation of p21ras Proteins-p21 ras (1-166) was expressed and purified as described previously (20). p21 ras C118S(1-166) was expressed and purified similarly.Generation of p21 ras C118S cDNA Constructs-Codon 118 of truncated (codons 1-166) Ha-ras cDNA was mutated from TGT (cysteine) to TCT (serine) using the polymerase chain reaction. The generated cDNA fragment was then sequenced (Sequenase) and cloned into the pATras bacterial expression vector. To generate full-length p21 ras C118S an NcoI/BamHI fragment (encoding residues 111-166 of the ras(1-166) mutant) was exchanged for a 0.8-kilobase fragment encoding residues 111-189 plus 3Ј-noncoding region and the coding junction sequenced. A BglII/BamHI fragment encoding full-length Ras(C118S) was then subcloned into the BamHI site of the pCDNA3 mammalian expression plasmid and orientation confirmed by BstXI digestion.CNBr Digestion and ESI-MS Analysis of p21 ras -One small crystal of CNBr (Fluka) was added to 100 pmol of p21 ras in 20 l of 0.1 N HCl in a 0.5-ml polypropylene tube. Digestion was carried out at room temperature for 10 min prior to analysis by ESI-MS. After 10 min, samples were directly electrosprayed into a Finnigan-MAT TSQ-700 triple quadrupole instrument for analysis of S-nitrosylation exactly as we d...

Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization (LC-ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon ␣-2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.