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One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR ζ ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon ζ ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of ζ chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein–phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein–phosphopeptide complex relative to the protein alone.

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.

Recent developments in instrumentation for ultraviolet resonance Raman (UVRR) spectroscopy and its application to proteins are reviewed. With excitation near the ∼ 195 nm amide π-π* transition strong enhancement is seen of the amide vibrational modes, particularly amide II, whose intensity is sensitive to secondary structure. With measurements at 200 and 192 nm one can calculate the fractions of α-helix, β sheet and unordered segments from the known cross sections of these structures. The proline imide II band at ∼ 1460 cm −1 is strongly enhanced with 218 nm excitation and its frequency can be used to monitor cis-trans isomerization. Aromatic residues give rise to strong UVRR bands. Phenylalanine (Phe) and tyrosine (Tyr) show scattering patterns typical of substituted benzenes: enhancement of vibronic modes, especially ν 8a and ν 8b in resonance with the quasi-forbidden L a transition (∼ 205 and ∼ 223 nm for Phe and Tyr), and of breathing modes in resonance with the allowed B a,b transitions (∼ 188 and ∼ 193 nm for Phe and Tyr). Tyrosine, and especially tyrosinate, also show strong enhancement of ν 8a and ν 8b in the B a,b -resonant region, behavior attributed to electronic mixing of L a with B a via the OH (or O − ) substituent. In proteins the Tyr ν 8a and ν 8b bands are most readily observed with 229 nm excitation, where the Phe contributions are minimal. The ν 8b frequency is sensitive to Tyr H-bonding and has been calibrated in terms of the H-bond strength. The Tyr 830/850 cm −1 Fermi doublet intensity ratio, which can be monitored at 200 nm is sensitive to H-bonding, but also to other environmental influences. With 218 nm excitation protein spectra are dominated by tryptophan (Trp) contributions. The 1340/1360 cm −1 Trp Fermi doublet is sensitive to solvent exposure, while the ∼ 880 cm −1 band frequency is sensitive to H-bonding. Histidine (His) excitation profiles show π-π* resonances at ∼ 218 and ∼ 204 nm. The UVRR bands are sensitive to protonation, but the enhancement is relatively weak, and the His bands are obscured in protein UVRR spectra by the other aromatic contributions. Applications of the UVRR technique to the filamentous virus fd are described.

Unlike its thiocyanate analogues, which involve the formation of the geminite [Pd{(C 6H 5) 2P(CH 2) 2-P(C 6H 5) 2}(SCN)(NCS)] and the di-N-bound complex [Pd{(C 6H 5) 2P(CH 2) 3P(C 6H 5) 2}(NCS) 2], the corresponding selenocyanate complexes contain only Sebound groups, as determined by the results of single crystal X-ray diffraction studies. Se-bonding is also maintained in the [Pd{(C 6H 5) 2PCH 2 o C 6H 4-CH 2P-(C 6H 5) 2}(SeCN) 2] complex, based on the results of IR studies. Only in the case of the (C 6H 5) 2PCH 2P(C 6H 5) 2 palladium(II) complexes, do both pseudohalides exhibit analogous bonding via their chalcogen atoms. The selenocyanate's bonding pattern is thus dominated by the soft/soft interaction afforded by Pd-SeCN bonding, remaining impervious to increasing steric demands of the diphosphine ligands.

The reaction, at 25 °C in methanol, between [Pd(SeCN)4]2− and bis(diphenylphosphino)methane (dpm) has been found to produce cyano(selenocyanato) [diphenyl(diphenylphosphinomethyl) phosphine selenide]palladium(II), [Pd(dpmSe)(CN)(SeCN)], wherein the cyanide group is trans to an Se atom which has been inserted into one PdP bond, and the selenocyanate group is trans to the unchanged diphenylphosphino group. The structure has been confirmed by the results of a single crystal X-ray diffraction study. The structural isomer, [Pd(dpm)- (SeCN)2], of the foregoing complex has also been prepared by the reaction of Pd(C2H3O2)2 with dpm, followed by reaction with KSeCN. Heating the [Pd(dpm)(SeCN)2] isomer converts it into [Pd- (dpmSe)(CN)(SeCN)]. A mechanism is proposed for the isomerization which involves an intramolecular selenium atom insertion.