Use of luminescent gunshot residues markers in forensic context-Part II.

Gunshot residues (GSR) are important physical evidence in firearm-related crimes. Recently developed non-toxic ammunition, however, requires a new methodology for its characterization. To overcome this drawback, the introduction of noncommercial luminescent markers in ammunition was proposed. These markers, synthesized and added to the gunpowder, presented as a versatile tool for GSR analysis, since they require UV radiation alone to visualize the luminescent GSR (LGSR). This has opened up new perspectives for understanding GSR behavior at a crime scene. This work aims to expand previous studies performed with the luminescent markers in forensic contexts, exploring four different important aspects related to GSR behavior. Using LGSR amount/dispersion and a series of blind tests with marked ammunition, we tried to (1) identify the shooter position; (2) estimate the shooting distance; (3) evaluate the influence of the pistol type on the LGSR distribution on the shooter's hands and guns; and (4) study the transference of LGSR by a chain of handshaking. For this purpose, a portable UV lamp (λ=254nm) and/or techniques such as video spectral comparator (VSC) and scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDS) were used to visualize and analyze the residues. As a result, the observation of the LGSR enabled accurate determination of the shooter position and the firing distance without any chemicals. Besides, the LGSR were visualized on different kinds of pistols, regardless of firing mechanism. And finally, this study evidenced transference of residues from shaking hands with the shooter, which should be carefully considered when interpreting the results of a GSR analysis.

Identification of ANFO: Use of luminescent taggants in post-blast residues.

This work describes the incorporation of luminescent taggants in cartridges of ammonium nitrate-fuel oil (ANFO) to allow easy identification and collection of post-blast residues. Metal-Organic Frameworks taggants proved to be appropriate for explosive marking when ANFO proportions were above 3.0wt.%. Lanthanide-based light emission enabled in situ retrieval of explosive residues as well as chemical identification, allowing the creation of a coding system using Energy Dispersive Spectroscopy (EDS). The proposed method will accelerate laboratory analysis and support forensic investigations, connecting evidence to suspects and/or to the explosive origin.

Use of luminescent gunshot residues markers in forensic context.

Chemical evaluation of gunshot residues (GSR) produced by non-toxic lead-free ammunition (NTA) has been a challenge to forensic analyses. Our group developed some luminescent markers specific to the detection of GSR. Here, we evaluated the performance of selected markers in experiments that mimic forensic context and/or routines in which luminescent characteristics would be very useful. We evaluated the influence of markers' addition on the bullet's speed, the rate of shot failure (i.e., when the cartridge case is not fully ejected and/or a new ammunition is not automatically replaced in the gun chamber) as a function of marker percentage, the possibility of collecting luminescent gunshot residue (LGSR) in unconventional locations (e.g. the shooters' nostrils), the LGSR lifetime after hand washing, the transfer of LGSR to objects handled by the shooter, and the dispersion of LGSR at the crime scene and on simulated victims. It was observed that high amounts of marker (10 wt%) cause high rates of failure on pistols, as well as a substantial decrease in bullet speed. However, the use of 2 wt% of marker minimizes these effects and allows LGSR detection, collection and analysis. Moreover, in all conditions tested, markers showed high performance and provided important information for forensic analyses. For instance, the LGSR particles were found on the floor, ranging from 0 to 9.4 m away from the shooter, on the door panel and seats after a car shooting experiment, and were found easily on a pig leg used to simulate a victim. When a selective tagging was done, it was possible to obtain positive or negative correlation between the victim and shooter. Additionally LGSR possesses a fairly long lifetime (9 h) and good resistance to hand washing (up to 16 washes).

Structure of Bacillus subtilis superoxide dismutase.

The sodA gene of Bacillus subtilis was expressed in Escherichia coli, purified and crystallized. The crystal structure of MnSOD was solved by molecular replacement with four dimers per asymmetric unit and refined to an R factor of 21.1% at 1.8 A resolution. The dimer structure is very similar to that of the related enzyme from B. anthracis. Larger structural differences were observed with the human MnSOD, which has one less helix in the helical domain and a longer loop between two beta-strands and also showed differences in three amino acids at the intersubunit interface in the dimer compared with the two bacterial MnSODs. These structural differences can be exploited in the design of drugs that selectively target the Bacillus enzymes.

Characterization of a naphthalene derivative inhibitor of retroviral integrases.

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.

Net charge center as the simplest model of a protein identifies up to 100% of active/binding site residues.

BACKGROUND: Electrostatic steering of ligands into active/binding sites is important for enzymatic catalysis and enhancing the affinity of metal-binding proteins. The net charge center of a protein, which describes the distribution of the charged residues in the protein subunit, has a specific location. As active sites in biological molecules are also located specifically, the biological implications of the model were examined by analyzing the relative locations of the net charge centers and the active/binding sites. MATERIAL/METHODS: Spatial structures from a set of 11 proteins with experimentally defined electrostatic steering residues and definitely known active site residues were analyzed. RESULTS: In 9 of 11 proteins, 70-100% of the active site residues belong to the residue cluster around the net charge center. On average, the charge center cluster identifies 78% of the active sites and 80% of the steering residues with few false positives. In several cases, comparison of proteins from different sources has shown that the differences in association rates correlate quantitatively with the relative locations of the net charge centers. CONCLUSIONS: In most of the proteins studied the net charge center is located close to the active site. Therefore, the active site in a novel enzyme or the binding site in a protein can be predicted solely from the three-dimensional structure of the protein. The residues forming clusters around the net charge center are often organized into continuous hydrophilic sequences, important for rapid and correct formation of the active site region during protein folding.

Structure of murine Tcl1 at 2.5 A resolution and implications for the TCL oncogene family.

Tcl1 and Mtcp1, members of the Tcl1 family, are implicated in T-cell prolymphocytic leukemia. The crystal structure of a dimer of murine Tcl1 has been determined at 2.5 A resolution with an R factor of 0.225. Murine Tcl1, human Tcl1 and Mtcp1 share very similar subunit structures, with RMS differences of 0.6 and 1.4 A for C(alpha) atoms, respectively, while the sequences share 50 and 36% identity, respectively. These structures fold into an eight-stranded beta-barrel of unique topology and high internal symmetry of 1.1-1.3 A for the two halves of human and murine Tcl1 and 1.7 A for Mtcp1, despite the low 12-13% sequence identity. The molecular surfaces of all three structures showed a common planar region which is likely to be involved in protein-protein interactions.

A functioning chimera of the cyclic nucleotide-binding domain from the bovine retinal rod ion channel and the DNA-binding domain from catabolite gene-activating protein.

The eukaryotic cyclic nucleotide-gated (CNG) ion channels are a family of large membrane proteins activated by cytoplasmic cGMP or cAMP. Their cyclic nucleotide-binding domain is structurally homologous with that of the catabolite gene-activator protein (CAP), a soluble Escherichia coli transcription factor. Differences in ligand activation among sensory channels suggest differences in the underlying molecular mechanisms of signal readout. To study the structural, functional, and conformational consequences of nucleotide binding, we fused the cyclic nucleotide-binding domain from the bovine retinal rod CNG channel alpha subunit (Bralpha) to the DNA-binding domain from CAP. The chimera forms a soluble dimer that binds both cGMP and cAMP with association constants of 3.7 x 10(4) M(-1) for [(3)H]cGMP and 3.1 x 10(4) M(-1) for [(3)H]cAMP. The binding of cAMP, but not cGMP, exposes a chymotrypsin cleavage site in the chimera at a position similar to the site in the CAP exposed by cAMP binding. At high cAMP concentrations, a biphasic pattern of cleavage is seen, suggesting that the low-affinity cAMP binding sites are also occupied. Cyclic AMP promotes specific binding to a DNA fragment encoding the lac operator region; the K(d) for the protein-DNA binding is approximately 200 nM, which is 2-fold higher than the K(d) for CAP under identical conditions. A 7 A crystal structure shows that the overall secondary and tertiary structure of Bralpha/CAP is the same as that of CAP with two cAMP molecules bound per dimer. The biochemical characterization of the chimera suggests it will be a useful system for testing hypotheses about channel activation, providing further insight into channel function.

Structural implications of drug-resistant mutants of HIV-1 protease: high-resolution crystal structures of the mutant protease/substrate analogue complexes.

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-A resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wild-type are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D.

Effects of different post-crystallization soaking conditions on the diffraction of Mtcp1 crystals.

The crystal structure of human Mtcp1 was determined at 2 A resolution after the X-ray diffraction limit was improved by post-crystallization soaking in 2.0 M ammonium sulfate for 1-5 months. The effects of varying the ammonium sulfate concentration and addition of polyethylene glycol to the soaking solution were examined in order to understand the phenomenon and to reduce the soaking time. Soaking the crystal for one week in a solution of 1.5 M ammonium sulfate and 2% PEG 3400 gave the desired improvement in diffraction quality. Therefore, different soaking conditions should be explored when crystals show disordered and low-resolution diffraction.

Intravirion display of a peptide corresponding to the dimer structure of protease attenuates HIV-1 replication.

Current treatment of HIV-1-infected individuals involves the administration of several drugs, all of which target either the reverse transcriptase or the protease activity of the virus. Unfortunately, the benefits of such treatments are compromised by the emergence of viruses exhibiting resistance to the drugs. This situation warrants new approaches for interfering with virus replication. Considering the activation of protease in the virus particles, a novel strategy to inhibit HIV-1 replication was tested targeting the dimerization domain of the protease. To test this idea, we have selected four residues from the C terminus of HIV-1 protease that map to the dimer interface region of the enzyme. We have exploited Vpr to display the peptides in the virus particles. The chimeric Vpr exhibited expression and virion incorporation similar to wildtype Vpr. The virus derived from the HIV-1 proviral DNA containing chimeric Vpr sequences registered a reduced level of replication in CEM and CEM X 174 cells in comparison with viruses containing wildtype Vpr. Similar results were observed in a single-round replication assay. These results suggest that the intravirion display of peptides targeting viral proteins is a powerful approach for developing antiviral agents and for dissecting the dynamic interactions between structural proteins during virus assembly and disassembly.

Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases.

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.

Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease.

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.

Structural model of human glucokinase in complex with glucose and ATP: implications for the mutants that cause hypo- and hyperglycemia.

Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Glucokinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interactions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the gamma-phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dramatic changes in the Km for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dramatically reduced the catalytic activity, as did a mutation that was predicted to disrupt an alpha-helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crystal structure for glucokinase, our models help rationalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activators and inhibitors.

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.

Molecular mechanics analysis of drug-resistant mutants of HIV protease.

Drug-resistant mutants of HIV-1 protease limit the long-term effectiveness of current anti-viral therapy. In order to study drug resistance, the wild-type HIV-1 protease and the mutants R8Q, V32I, M46I, V82A, V82I, V82F, I84V, V32I/I84V and M46I/I84V were modeled with the inhibitors saquinavir and indinavir using the program AMMP. A new screen term was introduced to reproduce more correctly the electron distribution of atoms. The atomic partial charge was represented as a delocalized charge distribution instead of a point charge. The calculated protease-saquinavir interaction energies showed the highly significant correlation of 0.79 with free energy differences derived from the measured inhibition constants for all 10 models. Three different protonation states of indinavir were evaluated. The best indinavir model included a sulfate and gave a correlation coefficient of 0.68 between the calculated interaction energies and free energies from inhibition constants for nine models. The exception was R8Q with indinavir, probably due to differences in the solvation energy. No significant correlation was found using the standard molecular mechanics terms. The incorporation of the new screen correction resulted in better prediction of the effects of inhibitors on resistant protease variants and has potential for selecting more effective inhibitors for resistant virus.

Improving the diffraction quality of MTCP-1 crystals by post-crystallization soaking.

Significant improvement in the resolution and quality of the X-ray diffraction of crystals of MTCP-1 protein was observed on post-crystallization soaking. The MTCP-1 crystals grown from 1.5 M ammonium sulfate diffracted to only 3.0 A resolution with some disorder in the diffraction. After post-crystallization soaking in a solution containing 2.0 M ammonium sulfate, the disorder was eliminated and diffraction extended to better than 2.0 A resolution. Both native and selenomethionine-enriched crystals demonstrated better diffraction after soaking for several months. This simple technique may be useful to improve the diffraction quality of protein crystals generally.

Drug-resistant HIV-1 proteases identify enzyme residues important for substrate selection and catalytic rate.

A series of mutations, first identified in protease inhibitor-resistant HIV-1 viral isolates, were introduced into HIV-1 PR as individual substitutions. Mutants containing R8K, V32I, V82T, I84V, G48V/L90M, or V82T/I84V substitutions were analyzed for differences in substrate preference and catalytic efficiency using a set of single amino acid substituted HIV-1 CA-NCa cleavage site peptides. All mutants exhibited wild-type preference for large hydrophobic residues, especially Phe, in the P1' substrate position. Only the R8K and V32I mutants showed significant differences in subsite selection compared to wild-type enzyme. In a parallel study, the individual mutations R10K, L12V, I44V, A60M, I71V, and I108V were introduced into RSV PR. These amino acid positions are structurally equivalent to Arg8, Leu10, Val32, Met46, Ile54, and Ile84 in HIV-1 PR, respectively, which mutate in drug-resistance. The RSV R10K substitution significantly altered substrate specificity and catalytic rate, compared to wild-type, in a manner similar to that of the HIV-1 R8K mutant. Crystal structures of the RSV PR R10K, I44V, I71V, and Il08V mutant enzymes presented here indicate that each of these substitutions has little effect on the overall structure of the respective enzymes. Taken together, these data provide an explanation for the reported in vivo predilection for selection of large hydrophobic residues in the P1' substrate position of second locus mutations in the Gag polyprotein PR cleavage sites. The data also suggest that the selection of resistant enzymes is not simply limited to loss of binding to inhibitor but affects other steps in proteolysis.