Photoaffinity labeling with [2‘-³²P]2N₃NADP⁺ and [³²P]2N₃NAD⁺ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP⁺-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorporation of 2N₃NAD⁺ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [³²P]2N₃NAD⁺ and [2‘-³²P]2N₃NADP⁺ showed saturation effects with apparent K_ds of 20 and 14 μM (±12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [³²P]8N₃ATP and [³²P]2N₃ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP⁺ followed by NAD⁺ and then ATP, indicating that photoinsertion with all the probes was within the NADP⁺ binding site. Isolation of [³²P]2N₃NAD⁺ and [2‘-³²P]2N₃NADP⁺ photolabeled peptides by use of immobilized boronate and immobilized Al³⁺ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile²⁴⁴-Arg²⁴⁹ and Leu¹²¹-Arg¹³³ (tryptic fragments) and Lys²⁴³-His²⁴⁸ and Leu¹²¹-His¹³⁵ (chymotryptic fragments). Trp¹²⁵ and Trp²⁴⁵ were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys²⁴³ and Lys¹²⁷. Sequence analysis of [³²P]8N₃ATP and [³²P]2N₃ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.

Human lung tryptase (HLT), a trypsin-like serine proteinase stored as an active enzyme in association with heparin in mast cell granules, is released into the extracellular environment when mast cells are activated. Tryptases are unusual in that they form tetramers and bind heparin. As there are no known endogenous tryptase inhibitors, loss of heparin and dissociation of the active tetrameric enzyme to inactive monomers has been proposed as the mechanism of control. Activity and intrinsic fluorescence were used to measure the stabilization of HLT by NaCl, glycerol, and heparin. At physiological salt concentrations in the absence of heparin, activity decayed rapidly (t_1/2 = 1−4 min at 37 °C) to an intermediate that could be immediately reactivated by heparin. But protein structural changes, as measured by intrinsic fluorescence, were much slower (t_1/2 = 16 min), indicating that the intermediate continued to exist as a tetramer that slowly changed to a monomer. HLT tetramers, either active or inactive, were stabilized by 2 M NaCl, 20% glycerol, and heparin. Maximum stabilization was obtained with approximately 1 mol of heparin per HLT subunit. Heparan sulfate also stabilized HLT activity and active HLT was bound to and recovered from cartilage. Subunits of the inactive intermediate appeared to be loosely associated as demonstrated by the rapid disappearance of the tetramer in gel filtration studies in 1 M NaCl (t_1/2 = 1.8 min), but the tetramer was stable in lower ionic strength buffers containing heparin. Fluorescence anisotropy measurements in the absence of heparin were also consistent with a slow (t_1/2 = 22 min) transition from tetramer to monomer, and native polyacrylamide gel electrophoresis provided additional evidence for a tetrameric intermediate. HLT monomers isolated by gel filtration were minimally active in the presence of heparin. These data show that heparin-free HLT rapidly converts to an “inactive”, loose tetrameric intermediate that can be reactivated with heparin or slowly dissociate to less active monomers and that tryptase released from mast cells is likely to remain active in association with heparin or other extracellular components. Thus, tryptase affinity for glycosaminoglycans and substrate specificity limitations are the primary factors controlling the proteolytic functions of these enzymes.

We describe a novel method of photo-cross-linking DNA-binding proteins to DNA employing psoralen as a tether. We apply this method for the interaction of T7 RNA polymerase to its promoter. The crystallographic model of T7 RNA polymerase shows a cleft formed by the palm, thumb, and fingers domains. It was proposed that template DNA binds in the cleft. Here we directly and positively identify, in solution, the cleft as the seat of template binding. We photo-cross-linked a 23 bp promoter DNA to T7 RNA polymerase. We then determined the masses of cross-linked tryptic peptides by mass spectrometry and analyzed their amino acid composition. The cross-linked peptides were projected on the crystal structure of T7 RNA polymerase. The peptides nicely decorated the back, front, and side wall of the cleft. In a previous work [Sastry et al. (1993) Biochemistry 32, 5526−5538] we used site-specific psoralen furan-side monoadducts for cross-linking DNAs to DNA-binding proteins. We cross-linked a single-stranded 12-mer oligonucleotide to T7 RNA polymerase. We isolated and purified a DNA cross-linked tryptic peptide. We then used mass spectrometry and amino acid composition analysis to identify the location of this peptide on the T7 RNA polymerase primary sequence. In the present work we have mapped this peptide on the 3-D structure of T7 RNA polymerase. This peptide maps in the fingers domain of the polymerase. On the basis of a comparison of the map positions of peptides that cross-linked to either promoter DNA or single-stranded oligo-DNA, we propose that different functional domains may be involved in binding of double-stranded promoter DNA and nonspecific single-stranded DNA. Whereas the cleft of the polymerase is the seat of double-stranded promoter binding, the fingers domain may be used by the polymerase to grab single-stranded DNA (or RNA) in a nonspecific manner. Alternatively, the single-stranded oligo binding site may be an RNA product-binding site during transcription. The photochemical techniques we have developed [Sastry et al. (1993) Biochemistry 32, 5526−5538; this work] can be applied to other DNA-protein complexes to map DNA−binding domains.

We report the structure of an NADPH:FMN oxidoreductase (flavin reductase P) that is involved in bioluminescence by providing reduced FMN to luciferase. The 1.8 Å crystal structure of flavin reductase P from Vibrio harveyi was solved by multiple isomorphous replacement and reveals that the enzyme is a unique dimer of interlocking subunits, with 9352 Ų of surface area buried in the dimer interface. Each subunit comprises two domains. The first domain consists of a four-stranded antiparallel β-sheet flanked by helices on either side. The second domain reaches out from one subunit and embraces the other subunit and is responsible for interlocking the two subunits. Our structure explains why flavin reductase P is specific for FMN as cofactor. FMN is recognized and tightly bound by a network of 16 hydrogen bonds, while steric considerations prevent the binding of FAD. A flexible loop containing a Lys and an Arg could account for the NADPH specificity. The structure reveals information about several aspects of the catalytic mechanism. For example, we show that the first step in catalysis, which is hydride transfer from C4 of NADPH to cofactor FMN, involves addition to the re face of the FMN, probably at the N5 position. The limited accessibility of the FMN binding pocket and the extensive FMN−protein hydrogen bond network are consistent with the observed ping-pong bisubstrate−biproduct reaction kinetics. Finally, we propose a model for how flavin reductase P might shuttle electrons between NADPH and luciferase.

Diaminopimelate dehydrogenase catalyzes the NADPH-dependent reduction of ammonia and l-2-amino-6-ketopimelate to form meso-diaminopimelate, the direct precursor of l-lysine in the bacterial lysine biosynthetic pathway. Since mammals lack this metabolic pathway, inhibitors of enzymes in this pathway may be useful as antibiotics or herbicides. Diaminopimelate dehydrogenase catalyzes the only oxidative deamination of an amino acid of d configuration and must additionally distinguish between two chiral amino acid centers on the same symmetric substrate. The Corynebacterium glutamicum enzyme has been cloned, expressed in Escherichia coli, and purified to homogeneity using standard biochemical procedures [Reddy, S. G., Scapin, G., & Blanchard, J. S. (1996) Proteins:  Structure, Funct. Genet. 25, 514−516]. The three-dimensional structure of the binary complex of diaminopimelate dehydrogenase with NADP⁺ has been solved using multiple isomorphous replacement procedures and noncrystallographic symmetry averaging. The resulting model has been refined against 2.2 Å diffraction data to a conventional crystallographic R-factor of 17.0%. Diaminopimelate dehydrogenase is a homodimer of structurally not identical subunits. Each subunit is composed of three domains. The N-terminal domain contains a modified dinucleotide binding domain, or Rossman fold (six central β-strands in a 213456 topology surrounded by five α-helices). The second domain contains two α-helices and three β-strands. This domain is referred to as the dimerization domain, since it is involved in forming the monomer−monomer interface of the dimer. The third or C-terminal domain is composed of six β-strands and five α-helices. The relative position of the N- and C-terminal domain in the two monomers is different, defining an open and a closed conformation that may represent the enzyme's binding and active state, respectively. In both monomers the nucleotide is bound in an extended conformation across the C-terminal portion of the β-sheet of the Rossman fold, with its C4 facing the C-terminal domain. In the closed conformer two molecules of acetate have been refined in this region, and we postulate that they define the DAP binding site. The structure of diaminopimelate dehydrogenase shows interesting similarities to the structure of glutamate dehydrogenase [Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., & Stillmann, T. J. (1992a) J. Mol. Biol. 228, 662−671] and leucine dehydrogenase [Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., & Rice, D. W. (1995) Structure 3, 693−705] and also resembles the structure of dihydrodipicolinate reductase [Scapin, G., Blanchard, J. S., & Sacchettini, J. C. (1995) Biochemistry 34, 3502−3512], the enzyme immediately preceding it in the diaminopimelic acid/lysine biosynthetic pathway.

The high-resolution solution structure of recombinant human basic fibroblast growth factor (FGF-2), a protein of 17.2 kDa that exhibits a variety of functions related to cell growth and differentiation, has been determined using three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry−simulated annealing using a total of 2865 experimental NMR restraints, consisting of 2486 approximate interproton distance restraints, 50 distance restraints for 25 backbone hydrogen bonds, and 329 torsion angle restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures for residues 29−152 is 0.43 ± 0.03 Å for the backbone atoms, 0.83 ± 0.05 Å for all atoms, and 0.51 ± 0.04 Å for all atoms excluding disordered side chains. The overall structure of FGF-2 consists of 11 extended antiparallel β-strands arranged in three groups of three or four strands connected by tight turns and loop regions creating a pseudo-3-fold symmetry. Two strands from each group come together to form a β-sheet barrel of six antiparallel β-strands. A helix-like structure was observed for residues 131−136, which is part of the heparin binding site (residues 128−138). The discovery of the helix-like region in the primary heparin binding site instead of the β-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin−FGF-2 interactions. A total of seven tightly bound water molecules were found in the FGF-2 structure, two of which are located in the heparin binding site. The first 28 N-terminal residues appear to be disordered, which is consistent with previous X-ray structures. A best fit superposition of the NMR structure of FGF-2 with the 1.9 Å resolution X-ray structure by Zhu et al. (1991) yields a backbone atomic rms difference of 0.94 Å, indicative of a close similarity between the NMR and X-ray structures.

In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2‘,3‘-epoxypropyl β-glycoside derivatives of the disaccharides, N,N‘-diacetylchitobiose [GlcNAc-β-(1→4)-GlcNAc] and N-acetyllactosamine [Gal-β-(1→4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 Å resolution. Under the conditions comprising 2.0 × 10^-3 M labeling reagent and 1.0 × 10^-5 M human lysozyme at pH 5.4, 37 °C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2‘,3‘-epoxypropyl β-glycoside derivatives of N,N‘-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N‘-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2‘,3‘-epoxypropyl β-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2‘,3‘-epoxypropyl β-glycoside of N-acetyllactosamine as compared to that of N,N‘-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.

The 42-residue β-(1−42) peptide is the major protein component of amyloid plaque cores in Alzheimer's disease. In aqueous solution at physiological pH, the synthetic β-(1−42) peptide readily aggregates and precipitates as oligomeric β-sheet structures, a process that occurs during amyloid formation in Alzheimer's disease. Using circular dichroism (CD) and ultraviolet spectroscopic techniques, we show that nicotine, a major component in cigarette smoke, inhibits amyloid formation by the β-(1−42) peptide. The related compound cotinine, the major metabolite of nicotine in humans, also slows down amyloid formation, but to a lesser extent than nicotine. In contrast, control substances pyridine and N-methylpyrrolidine accelerate the aggregation process. Nuclear magnetic resonance (NMR) studies demonstrate that nicotine binds to the 1−28 peptide region when folded in an α-helical conformation. On the basis of chemical shift data, the binding primarily involves the N-CH₃ and 5‘CH₂ pyrrolidine moieties of nicotine and the histidine residues of the peptide. The binding is in fast exchange, as shown by single averaged NMR peaks and the lack of nuclear Overhauser enhancement data between nicotine and the peptide in two-dimensional NOESY spectra. A mechanism is proposed, whereby nicotine retards amyloidosis by preventing an α-helix → β-sheet conformational transformation that is important in the pathogenesis of Alzheimer's disease.

The serine protease, subtilisin BPN‘, was engineered to cleave proteins after tribasic sequences in a manner that resembles the substrate specificity of furin, one of the mammalian subtilisin homologs that processes prohormones. As a starting point we used a double mutant of subtilisin BPN‘ (N62D/G166D) that showed substantial preference for cleaving after sequences having consecutive dibasic residues (namely, at the P1 and P2 substrate positions) [Ballinger et al. (1995) Biochemistry 34, 13312−13319]. Additional specificity for basic residues was engineered at the P4 position by introducing subtilisin-to-furin substitutions at three hydrophobic residues that composed the S4 subsite (Y104, I107, and L126). Initial attempts to incorporate a Y104D or I107E mutation or the Y104D/I107E double mutation into the dibasic specific enzyme failed to generate the processed enzyme. The problem was traced to the inability of the mutant prosubtilisins to process themselves and fold correctly. Replacing the natural processing site sequence (AHAY) with a good furin substrate sequence (RHKR) resulted in expression of the triple subtilisin mutant (N62D/Y104D/G166D) we call “furilisin”. Furilisin hydrolyzes synthetic tribasic substrates (succinyl-RAKR-pNA or succinyl-KAKR-pNA) with high catalytic efficiency (k_cat/K_m >3 × 10⁵ M^-1 s^-1) and discriminates in favor of Arg versus Ala at the P4 position by a factor of 360. The overall specificity change versus the wild-type enzyme was dramatic. For example, succinyl-RAKR-pNA was cleaved ∼60 000 times faster than succinyl-AAPF-pNA, a good substrate for wild-type subtilisin. Similarly, furilisin was inhibited (K_i* = 29 nM) by a variant of the turkey ovomucoid third domain inhibitor that contained an engineered furin substrate site (RCKR↓) [Lu et al. (1993) J. Biol. Chem. 268, 14583−14585] and not by one having a good wild-type subtilisin substrate sequence (ACTL↓). Interestingly, the extreme changes in substrate specificity resulted from substantial synergy between the engineered subsites. These studies provide a basic example of how to manipulate substrate specificity in a modular fashion, thereby creating an engineered enzyme that may be useful as a protein processing tool.

The iron responsive element (IRE) is a conserved RNA structure that is found in the 5‘ UTR of ferritin mRNA and in the 3‘ UTR of transferrin receptor mRNA. It is the binding site of the iron responsive protein (IRP), and the interaction is part of the regulation of cellular iron metabolism. The IRE six-nucleotide hairpin loop, 5‘C₁A₂G₃U₄G₅N₆, is conserved in sequence, and mutations have shown that it is required for IRP binding. On the basis of the thermodynamic and NMR experiments utilized here, the IRE loop structure 5‘C₁A₂G₃U₄G₅C₆, is described in detail. Measurements of loop stability show that it has 2.9 kcal/mol more free energy than predicted. NMR data suggest that there is hydrogen bonding between C1 and G5 in a tertiary interaction across the loop. A model structure, produced by MC-SYM/energy minimization, illustrates the conformational flexibility of U4 and C6, which appear to exhibit considerable local motion in solution. NMR data indicate that the position of G3 is not well defined, leading to two families of loop structures.

The structures in solution of eight oligonucleotide duplexes each containing either zero, one, or two 5-fluorodeoxyuridine (FdUrd) or 5-fluorouridine (FUrd) nucleosides were determined by the combined use of NMR spectroscopy, restrained molecular dynamics, and full relaxation matrix refinement to determine how FdUrd and FUrd substitution affects the structure of duplex DNA and RNA and to establish whether structural differences due to FdUrd and FUrd substitution in nucleic acids may be responsible, in part, for the biological effects of the anticancer drug 5-fluorouracil (FUra). The nucleic acid directed effects of FUra include induction of single-strand breaks in duplex DNA and altered processing of pre-mRNA and rRNA. Four self-complementary oligodeoxyribonucleotide sequences were prepared and studied as duplexes in aqueous solution:  (5‘ dGCGAAUUCGC)₂, (5‘ dGCGAAUFCGC)₂, (5‘ dGCGAAFUCGC)₂, and (5‘ dGCGAAFFCGC)₂. The corresponding oligoribonucleotide sequences (5‘ rGCGAAUUCGC)₂, (5‘ rGCGAAUFCGC)₂, (5‘ rGCGAAFUCGC)₂, and (5‘ rGCGAAFFCGC)₂ were also prepared and studied. The helical parameters for the structures of these eight duplexes were analyzed to determine how substitution of FdUrd and FUrd affects the three-dimensional structures of duplex DNA and RNA. FdUrd substitution affects the base roll angle at the site of FdUrd substitution, causing the helical axis of FdUrd-substituted DNA duplexes to be bent compared to the nonsubstituted duplex. A-FUrd base pairs show substantial RMS deviations from A-Urd base pairs in all three of the RNA duplexes substituted with FUrd. Bending of the helical axis due to FdUrd substitution may contribute to the occurrence of single-strand breaks in duplex DNA while the altered structures of A-FUrd base pairs may affect RNA−RNA and RNA−protein recognition.

The structure of the specific phosphate binding loop (P-loop) of bovine protein tyrosine phosphatase (BPTP) is very similar to that present in high M_r PTPases. Site-directed mutagenesis was used to explore the role of several conserved residues involved in forming the P-loop of BPTP. Thus, Ser-19 and Ser-43 were individually mutated to alanines, and Asn-15 was mutated to alanine and glutamine. The ¹H NMR spectra of the mutants showed good conservation of global secondary structure when compared to wild-type enzyme. Kinetic measurements revealed that only S19A and N15A had substantially altered catalytic activities toward p-nitrophenyl phosphate at pH 5.0, with both mutants exhibiting V_max values that were 0.25−0.33% of wild-type enzyme. Further kinetic analyses of the N15A and S19A mutants were performed using phosphomonoester substrates with varied phenolic leaving groups. For S19A, the slope of the correlation between V_max and the substrate leaving group pK_a was significantly altered, consistent with a change of the rate-determining step from dephosphorylation to phosphorylation. This was confirmed by partitioning experiments employing methanol as an alternative nucleophile in the dephosphorylation step. Thus, mutating Ser-19 to alanine reduced the efficiency of nucleophilic attack by Cys-12. It is concluded that Ser-19 acts to facilitate the ionization and orientation of Cys-12 for optimal reaction as a nucleophile and as a leaving group. It also appears that Asn-15, Ser-19, His-72, and to a lesser extent Ser-43 serve structural functions that allow the active site to adopt an optimal geometry for phosphate binding. The Asn-15 to Ala mutation appears to disrupt the hydrogen-bonding network, with an accompanying alteration of the geometry of the P-loop. These conclusions are also consistent with changes in the stability of the respective proteins, as measured by urea denaturation.

We have generated mutants of cytochrome b₅₆₂ in which the histidine ligand to the heme iron (His102) has been replaced by a methionine. The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation state and solution pH. We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions. Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation. In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated. We cannot identify what, if anything, provides the other axial ligand. A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state. With a pK_a of 7.1 at 20 °C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant. Our spectroscopic data prove that the low-spin species is bis-methionine coordinated. The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8. The electrochemistry of these proteins is complicated by coupled coordination-state changes. Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.

Previous work has shown that, in variants of cytochrome b₅₆₂ containing the H102M mutation, methionine residues provide both axial ligands to the heme iron. NMR spectroscopic studies of such bis-methionine-coordinated cytochrome have not previously been feasible, since the only other cytochrome with such a ligand arrangement, bacterioferritin, is too large to be studied by current NMR methods. The present work provides the first NMR characterization of 6-coordinate, bis-methionine-ligated heme centers in both ferrous and ferric oxidation states. We have used one and two dimensional, homonuclear NMR spectroscopy to assign the proton resonances of the heme group and ligand side chains in the reduced, cytochrome b₅₆₂ variants, H102M and covR98C/H102M. The latter protein has heme covalently attached to the protein, and our results prove that the covalent linkage is a c-type thioether bond formed between the cysteine at residue 98 and the heme 2-vinyl group. Spectra of the ferrous H102M variant are consistent with the presence of two species differing in the orientation of the heme in the protein. We have interpreted results from NOESY experiments on the ferrous covR98C/H102M protein in terms of the conformation of the two methionine side chains, and we present a model for the structure of the heme ligand arrangement. The Met7 side chain adopts an extended conformation almost identical to that observed in the wild type protein with R stereochemistry at the chiral sulfur ligand. The Met102 side chain has a different, buckled side chain conformation and has S stereochemistry at the chiral center. Our NMR derived model is consistent with the spectroscopic data presented in the previous paper. Studies on the ferric forms of these proteins confirm that the double variant at low pH has a “stable” bis-methionine ligation arrangement, but that it is a thermal mixture of species with differing spin states. No hyperfine coupled proton resonances can be identified in spectra of the high-spin forms of either of these proteins.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T₁ (rd-RNase T₁) and mixed disulfide between glutathione and reduced RNase T₁ (GS-RNase T₁) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding experiments were performed in the presence of different absolute concentrations of glutathione species while keeping the redox potential fixed, in order to evaluate the effect of the glutathione concentration on the distribution of the refolding intermediates. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T₁ which occurs via the reiteration of two steps:  (i) formation of a species containing both mixed disulfides with glutathione and free protein thiols, and (ii) formation of an intramolecular disulfide via thiol−disulfide interchange reaction betwen them. Refolding of rd-RNase T₁ and GS-RNase T₁ was also performed in the presence of protein disulfide isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. Refolding reactions carried out at different absolute concentrations of glutathione proved that GSH and/or GSSG participate directly in the reaction catalyzed by PDI. On the basis of these experiments and previous results on the refolding of RNase A [Torella, C., Ruoppolo, M., Marino, G., & Pucci, P. (1994) FEBS Lett. 352, 301−306], a hypothesis of a general pathway for folding of S−S containing proteins is proposed.

MRP is a recently described ATP-binding cassette transporter that confers cellular resistance to natural product cytotoxic drugs. To examine the biochemical activity and cellular physiology of this transporter, we isolated the murine MRP homologue and analyzed its in vitro substrate specificity. Murine MRP transcript is widely expressed in tissues and encodes a protein of 1528 amino acids that is 88% identical to its human homologue. Hydropathy analysis indicated that murine and human MRP, the yeast cadmium resistance transporter and the sulfonylurea receptor share a conserved topology distinguished from P-glycoprotein and the cystic fibrosis conductance regulator by an N-terminal hydrophobic region that contains several potential transmembrane domains. Drug uptake assays performed with membrane vesicles prepared from NIH3T3 cells transfected with a murine MRP expression vector revealed ATP-dependent transport for the natural product cytotoxic drugs daunorubicin and vincristine, as well as for the glutathione S-conjugates leukotriene C₄ and azidophenacyl-S-glutathione. Drug transport was osmotically sensitive and saturable with regard to drug and ATP concentrations, with K_m values of 19 μM, 19 μM, 26 nM, 17 μM, and 77 μM for daunorubicin, vincristine, leukotriene C₄, APA-SG, and ATP, respectively. Consistent with broad substrate specificity, the drug glutathione conjugate APA-SG, oxidized glutathione, the LTD₄ antagonist MK571, arsenate, and genistein were competitive inhibitors of daunorubicin transport, with K_i values of 32 μM, 25 μM, 1.9 μM, 108 μM, and 23 μM, respectively. This study demonstrates that the substrate specificity of murine MRP is quite broad and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression pattern of murine MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that it represents a widespread and versatile cellular defense mechanism.

The growth and shortening of microtubules in dynamic instability is known to be modulated by microtubule-associated proteins (MAPs). A full understanding of the mechanism of dynamic instability requires that one distinguish which of its aspects are mediated by microtubule-associated proteins (even in small residual concentrations) and which are intrinsic properties of the tubulin lattice itself. This paper addresses two of those aspects:  whether MAPs cause the rescue events of dynamic instability (i.e., the transitions from shortening to growth) and whether MAPs are responsible for the marked variability of the rates at which microtubules grow and shorten. Very pure tubulin was prepared by sequential chromatographies on phosphocellulose and DEAE-Sephadex. Analysis by electrophoresis and immunoblotting showed it to be essentially MAP-free; it contained fewer than one MAP molecule per 10 000 tubulin dimers. When its dynamic instability was studied by video-DIC microscopy, rescues were found to occur at a mean frequency of one per 4 μm of shortening. Variability of rates of growth and shortening, which is observed on the length scale of a few micrometers, was not changed by removal of MAPs. Because the mean distance between bound MAP molecules was calculated to be greater than 14 μm in these experiments, it is concluded that they cannot cause either rescue or variability of rates.

A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-dihydrodiols to yield o-quinones, a reaction catalyzed by dihydrodiol dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-diol) was oxidized by this route to yield benzo[a]pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [³H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 ± 17 adducts/10⁶ base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes treated with either BP-diol or BPQ were found to produce large quantities of superoxide anion radical (O₂^•-). The amount of O₂^•- generated by BP-diol was blocked by DD inhibitors. These data suggest that by diverting BP-diol to BPQ reactive oxygen species (ROS) were generated which caused DNA fragmentation. The ability of BPQ to cause DNA strand scission was further studied using supercoiled φX174 DNA. It was found that BPQ caused concentration-dependent (0.05−10 μM) strand scission in the presence of 1 mM NADPH (which promoted redox-cycling) provided CuCl₂ (10 μM) was present. Complete destruction of the DNA was observed using 10 μM BPQ. This strand scission was prevented by catalase and hydroxyl radical scavengers but not by superoxide dismutase. These data indicate that ROS were responsible for the destruction of the DNA. Using 20 μM (±)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] only single nicks in the DNA were observed indicating that BPQ was the more potent chemical nuclease. BPQ was also found to be a direct-acting mutagen in the Ames test using Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA104, but was 10−5500-fold less efficient as a mutagen than (±)-anti-BPDE. Our data indicate that DD suppresses the mutagenicity of (±)-anti-BPDE by producing BPQ, but in doing so a potent chemical nuclease is produced which causes extensive DNA fragmentation via the generation of ROS.

Site-directed mutagenesis was used to investigate the mechanism of electron and proton transfer in the ubiquinol oxidase, cytochrome bo₃, from Escherichia coli. The reaction between the fully reduced form of the enzyme and dioxygen was studied using the flow−flash method. After rapid mixing of CO-bound enzyme with an O₂-containing solution, CO was photodissociated, and the subsequent electron- and proton-transfer reactions were measured spectrophotometrically, the latter using a pH-indicator dye. In the wild-type, pure bo₃ enzyme, without bound quinones, we observed a single kinetic phase with a rate constant of about 2.4 × 10⁴ s^-1, associated with formation of the ferryl oxygen intermediate, followed by proton uptake from solution with a rate constant of about 1.2 × 10⁴ s^-1. Enzyme in which heme o instead of heme b was incorporated into the low-spin site displayed a slower ferryl formation with a rate constant of about 3.6 × 10³ s^-1. Upon replacement of the acidic residue glutamate 286 in helix VI of subunit I with a nonprotonatable residue, electron transfer was slightly accelerated, and proton uptake was impaired. Mutations of other residues in the vicinity of E286 also resulted in a dramatic decrease of proton uptake, suggesting that the environment of this residue is important for efficient proton transfer. In the closely related cytochrome aa₃ from P. denitrificans, the corresponding residue (E278) has been suggested to be part of a proton-transfer pathway [Iwata, S., Ostermeier, C., Ludwig, B., & Michel, H. (1995) Nature 376, 660−669]. The results are discussed in terms of a model for electron−proton coupling during dioxygen reduction.

A structural parameterization of the folding energetics has been used to predict the effect of single amino acid mutations at exposed locations in α-helices. The results have been used to derive a structure-based thermodynamic scale of α-helix propensities for amino acids. The structure-based thermodynamic analysis was performed for four different systems for which structural and experimental thermodynamic data are available:  T4 lysozyme [Blaber et al. (1994) J. Mol. Biol. 235, 600−624], barnase [Horovitz et al. (1992) J. Mol. Biol. 227, 560−568], a synthetic leucine zipper [O'Neil & Degrado (1990) Science 250, 646−651], and a synthetic peptide [Lyu et al. (1990) Science 250, 669−673]. These studies have permitted the optimization of the set of solvent-accessible surface areas (ASA) for all amino acids in the unfolded state. It is shown that a single set of structure/thermodynamic parameters accounts well for all the experimental data sets of helix propensities. For T4 lysozyme, the average value of the absolute difference between predicted and experimental ΔG values is 0.09 kcal/mol, for barnase 0.14 kcal/mol, for the synthetic coiled-coil 0.11 kcal/mol, and for the synthetic peptide 0.08 kcal/mol. In addition, this approach predicts well the overall stability of the proteins and rationalizes the differences in α-helix propensities between amino acids. The excellent agreement observed between predicted and experimental ΔG values for all amino acids validates the use of this structural parameterization in free energy calculations for folding or binding.

The interactions between RNA structures, such as RRE in the HIV-1 genome, and proteins, such as Rev of HIV-1, are essential for efficient viral replication. Compounds that bind specifically to such RNAs and disrupt their protein complexes offer a novel mechanism for inhibition of replication of the virus. As a step in this approach, we have designed and characterized a series of synthetic diphenylfuran cations that selectively inhibit Rev binding to RRE. Fluorescence titrations and gel band-shift results indicate that the diphenylfurans bind to RRE and inhibit Rev complex formation in a structure-dependent manner. The derivative with the greatest affinity for RRE has an association constant of greater than 10⁷ M^-1 and inhibits formation of the Rev−RRE complex at concentrations below 1 μM. It binds to RRE considerably more strongly than it binds to simple RNA duplexes. Spectral changes and energy transfer results on complex formation suggest that the compound has a nonclassical intercalation binding mode. CD studies with modified RRE hairpins indicate that the active diphenylfurans bind at the structured internal loop of RRE and cause a conformational change. The most active diphenylfurans are tetracations that appear to bind to RRE by a threading intercalation mode and cause a conformational change in the RNA that is essential for inhibition of Rev complex formation with RRE.

Two synthetic peptides corresponding to sequences in HIV-1_LAI gp41, T21 (aa 558−595) and T20 (aa 643−678), are strong inhibitors of HIV-1 viral fusion, having EC₅₀ values of 1 μg/mL and 1 ng/mL, respectively. Previous work suggested that T21 forms a coiled-coil structure in PBS solution, while T20 is primarily nonhelical, and that the inhibitory action of these peptides occurs after the interaction between the viral gp120 protein and the cellular CD4 receptor [Wild, C. T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., Matthews, T. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9770 and references therein]. The current study uses sedimentation equilibrium (SE), circular dichroism (CD), and viral-fusion assays to quantitatively investigate peptide structure and peptide−peptide interactions. SE analyses of T21 (1−100 μM) indicate that the peptide self-associates via a monomer/dimer/tetramer equilibrium; in addition, T20 is monomeric in the range of 1−10 μM and exhibits a complicated monomer/tetramer equilibrium between 20 and 100 μM. Singular value decomposition analyses of the CD spectra of T21 and T20 indicate that the helical content of these peptides in PBS solution is 90% and 20%, respectively. A structural interaction between the two peptides is detected by CD at several concentration ratios of T20:T21. These experiments emphasize that T20 interacts specifically with the tetrameric form of T21. Truncated forms of T20 also exhibit structural interactions with T21 at varying concentration ratios. The ability of T20 and the truncated peptides to interact structurally with tetrameric T21 correlates with antiviral activity. Implications of these findings are discussed in terms of proposed mechanisms of membrane fusion inhibition and the structural changes which occur in gp41 during membrane fusion.