Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.     

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.     

A novel soluble tissue factor variant with an altered factor VIIa binding interface

Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa). To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein. TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa. Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence. Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface. The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58. Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation. Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.     

Tyrosine quenching of tryptophan phosphorescence in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Tyrosine is known to quench the phosphorescence of free tryptophan derivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demonstrated. This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by comparing the phosphorescence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, is replaced with phenylalanine or Y283 is replaced by valine. Phosphorescence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp84-->Phe) mutant proteins, is already quenched in viscous low-temperature solutions, before the onset of major structural fluctuations in the macromolecule, an anomalous quenching that is abolished with the mutation Y283V (Tyr283-->Val). In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 micros to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction with Y283. Tyr quenching of W310 exhibits a strong temperature dependence, with a rate constant kq = 0.1 s(-1) at 140 K and 2 x 10(4) s(-1) at 293 K. Comparison between thermal quenching profiles of the W84F mutant in solution and in the dry state, where protein flexibility is drastically reduced, shows that the activation energy of the quenching reaction is rather small, Ea < or = 0.17 kcal mol(-1), and that, on the contrary, structural fluctuations play an important role on the effectiveness of Tyr quenching. Various putative quenching mechanisms are examined, and the conclusion, based on the present results as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an excited-state triplet exciplex.     

Compact thermally-denatured state of a staphylococcal nuclease mutant from resonance energy transfer measurements

Thermal denaturation of a staphylococcal nuclease mutant K78C, where lysine 78 is replaced by cysteine, was studied by circular dichroism (CD) and resonance energy transfer. CD spectra suggest that residual structures remain in the denatured state. Steady-state energy transfer from intrinsic tyrosines to a single and intrinsic tryptophan was measured at different temperatures. In the thermally-denatured state of K78C, there is still a substantial degree of energy transfer from tyrosine(s) to tryptophan, indicating residual structures in the denatured state. The cysteine residue in mutant K78C was labeled with a cysteine specific probe IAEDANS. Fluorescence decays of the tryptophan were measured to estimate distance distributions between Trp 140 and IAEDANS at position 78. Measurements were done as a function of temperature from 4 degrees C (native) to 65 degrees C (denatured) both with and without Ca2+ and inhibitor pdTp. Below 30 degrees C, the apparent distance distribution of both the ligand-free nuclease and the enzyme with bound pdTp can be adequately described by a Gaussian model. Above 40 degrees C, where the ligand-free nuclease but not the ternary complex begins to denature, two different populations are required to fit the data both with and without pdTp. One population has a compact structure and the other has an expanded structure. As temperature rises, the population of the expanded structure increases. At the highest temperature, the non-native compact structure is still the major form (60 to 70%). The overall thermally-denatured states of staphylococcal nuclease mutant K78C in the absence and presence of ligands are thus compact and heterogeneous.     

Influence of bulky side chains of amino acids on the solution conformation of peptide fragment (81-92) derivatives of CD4, TYICEVEDQKEE, as studied by NMR spectroscopy and molecular modeling

Solution conformation of CD4 fragment 81-92 TYICEVEDQKEE and two of its benzylated analogues was determined by two-dimensional 1H NMR spectroscopy, distance geometry and simulated annealing techniques. The structures of both benzylated derivatives are similar but are distinct from that of wild-type dodecapeptide. It is concluded from structural analysis that bulky side chain(s) of amino acid(s) at an appropriate position can have a marked effect on the conformation and thus the functions of a peptide.     

Evidence for the involvement of arginine 462 and the flanking sequence of human C4 beta-chain in mediating C5 binding to the C4b subcomponent of the classical complement pathway C5 convertase

Replacement of human C4 beta-chain residue arginine 458 by tryptophan, a substitution that occurs naturally in the hemolytically inactive A6 allotype of C4, totally abrogates the molecule's ability to act as a C5 binding subunit of the classical pathway C5 convertase. Hydropathy plots predict R458 to be within a hydrophilic segment extending from residue 455 to 469 and having the sequence SIERPDSRPPRVGDT. To further assess the potential involvement of this segment in the C5 binding function of C4, we have engineered "ala-scan" mutants through this segment, concentrating predominantly on charged residues, and analyzed their functional profiles. C4B isotype mutant proteins S455A (0.7), E457A (1.1), R458A (0.3), D460A (0.2), R462A (0.0), R465A (0.6), and D468A (0.3) displayed the relative to wild-type hemolytic activities indicated in the parentheses. In all cases, the hemolytic defect was accounted for solely at the C5 convertase stage. The total absence of C5 binding activity in the R462A mutant suggests a requirement for the guanidinium group per se, because mutants with a charge-conservative lysine or a relatively isosteric methionine at this position were also completely inactive. In contrast, the inactivity of the C4A6-like R458W mutant is probably caused by the intolerance of tryptophan in a hydrophilic segment, as substitution of R458 by alanine or methionine yielded recombinant molecules that retained 30% and 60% of wild-type hemolytic activity, respectively. Taken together, the mutagenesis results strongly imply that residues in the 455-469 segment contribute to the C5 binding site in C4; however, the conformational context of the segment appears to be crucial, as a synthetic peptide corresponding to this segment displayed no ability to inhibit C5 binding to surface-bound C4b.     

Midazolam premedication increases sedation but does not prolong discharge times after brief outpatient general anesthesia for laparoscopic tubal sterilization

Oligonucleotide-directed mutagenesis was utilized to investigate the requirement of tryptophan residues located in the N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor for the ability to bind its ligand and to induce cAMP generation. W39, W72, W87, W91, W110, and W120 were mutated into alanine. Two of the six tryptophan residues, W72 and W110, are highly conserved within the receptor subfamily. After transfection of mutated cDNAs in COS-7 or CHL cells, it appeared that mutant W87 A bound [125I] GLP-1 with the same affinity as wild-type receptor and induced signal transduction to a comparable extent. In contrast, mutant receptors W39A, W72A, W91A, W110A, and W120A lost the ability to bind [125I] GLP-1. Because all mutated receptor cDNAs were transcribed on RNA level (Northern blot) and the receptor proteins were expressed at the plasma membrane level (Western blot), it is concluded that with the exception of W87 all trytophan residues are essential for receptor ligand interaction. This indicates the significance of hydrophobic interactions within the N-terminal domain of the GLP-1 receptor.     

Tryptophan-containing mutant of human (group IIa) secreted phospholipase A2 has a dramatically increased ability to hydrolyze phosphatidylcholine vesicles and cell membranes

Human nonpancreatic (group IIa) secreted phospholipase A2 (human sPLA2) is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated. It is probable that the enzyme normally acts as an acute-phase protein whose function is to facilitate the removal of infectious organisms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) vesicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mammalian pancreatic enzymes show modest activity with such interfaces and contain a unique tryptophan at position 3, which is part of the presumptive interfacial binding surface of these enzymes. Human sPLA2 does not contain tryptophan. The amphiphilic indole side chain of tryptophan is noted for its ability to penetrate the lipid interface of membranes, and tryptophan residues appear to be associated with the ability of lipases and phospholipases A2 to bind to and hydrolyze such interfaces. We have investigated in detail the properties of a V3W mutant of human sPLA2, which has a unique tryptophan on the interfacial binding surface of this enzyme. Although this enzyme shows a modest ( approximately 50%) reduction in activity when anionic substrates are used under standard assay conditions, the activity of the enzyme on phosphatidylcholine vesicles and cell membranes is dramatically increased compared with human sPLA2. This is particularly the case with small unilamellar vesicles of PC, where activity is enhanced over 250-fold compared to the almost zero activity expressed by human sPLA2. This enhanced activity is best explained by increased interfacial binding and activation of the V3W mutant and is not due to enhanced active-site binding and hydrolysis. The results highlight the important role that tryptophan residues can play in interfacial binding, particularly to condensed zwitterionic interfaces. The interfacial characteristics of the mutant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.     

Universal fungus-specific primer systems and group-specific hybridization oligonucleotides for 18S rDNA

We designed two primer systems that amplify a fragment of the gene coding for the small ribosomal subunit (18S rRNA). A broadly reactive, yet fungus-specific, primer cocktail comprises two previously published primers, TR1 and TR2, which specifically amplify dermatophytes, and two newly designed primers, CA1 and AF2, which specifically amplify Candida and Aspergillus respectively. This primer cocktail amplifies a DNA fragment of approximately 578 basepairs (bp) in length (from position 838 to 1415), which contains variable, possibly species-specific regions (V5, partly V7). Another newly designed primer, UF1 (universal fungal primer 1), along with the eukaryotic primer S3 amplifies a 926-bp fragment (from position 263 to 1188) that includes the variable regions V3, V4 and V5. Both primer systems amplified DNA from Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, Fusarium oxysporum and Trichophyton mentagrophytes, but not the DNA from Prototheca zopfii, Escherichia coli or humans. The previously published oligonucleotides TR and HC, which are specific for dermatophytes and Histoplasma respectively, and the newly designed group-specific oligonucleotides, CA and AF, hybridized with T. mentagrophytes, Histoplasma capsulatum, C. albicans and A. fumigatus respectively, but not with the other six fungi or with the three controls.     

Long-term bacterial profile of refrigerated ground beef made from carcass tissue, experimentally contaminated with pathogens and spoilage bacteria after hot water, alkaline, or organic acid washes

The effects of 2% (vol/vol) lactic acid (LA), 2% (vol/vol) acetic acid (AA), 12% (wt/vol) trisodium phosphate (TSP), 72 degrees C water (HW), and 32 degrees C water (W) washes on bacterial populations which were introduced onto beef carcass surfaces after wash treatments were determined up to 21 days of storage at 4 degrees C of packaged ground beef prepared from the treated and inoculated carcasses. Beef carcass necks were collected from cattle immediately after harvest and subjected to the above treatments or left untreated (control). Neck meat was then inoculated with low levels (ca. <2 log10) of Listeria innocua, Salmonella typhimurium, Escherichia coli O157:H7, and Clostridium sporogenes contained in a bovine fecal cocktail. In general, growth of these four bacteria, aerobic bacteria, lactic acid bacteria, and pseudomonads was suppressed or not observed in the ground beef when LA, AA, or TSP treatments were used as compared to the untreated control. HW or W washes offered little suppression of growth of pathogens during subsequent storage of ground beef when these bacteria were introduced onto beef tissue posttreatment. Of the treatments used, a final LA or AA wash during the processing of beef carcasses offers the best residual efficacy for suppression of pathogen proliferation in ground beef during long-term refrigerated storage or short-term abusive temperature storage if these bacteria contaminate the carcass immediately after carcass processing.     

Monitoring the sizes of denatured ensembles of staphylococcal nuclease proteins: implications regarding m values, intermediates, and thermodynamics

Fluorescence and size-exclusion chromatography (SEC) are used to monitor urea denaturation of wild-type staphylococcal nuclease (SN) as well as the m+ and m- mutants A69T and V66W, respectively. It is found that the SEC partition coefficient, 1/Kd, is directly proportional to the Stokes radii of proteins. From the Stokes radii, the denatured ensembles of the three proteins are found to be highly compact in the limit of low urea concentration and expand significantly with increasing urea concentration. The m values from fluorescence-detected denaturation of the SN proteins are generally considered to reflect the relative sizes of denatured ensembles. However, the rank order of m values of the SN proteins studied do not correspond to the rank order of denatured ensemble sizes detected by 1/Kd, suggesting that m values reflect more than just surface area increases on denaturation. SEC provides two complementary ways to demonstrate the existence of intermediates in urea denaturation and illustrates that V66W undergoes a three-state transition. Fluorescence-detected urea denaturations of A69T and wt SN do not correspond with 1/Kd-detected denaturation profiles, a result that would ordinarily mean that the transitions are non-two-state. However, this interpretation fails to recognize the rapidly changing size and thermodynamic character of the denatured ensembles of these proteins both within and outside of the transition zone. The implications of the changing sizes and thermodynamic character of the denatured ensembles for SN proteins are manifold, requiring a reconsideration of the thermodynamics of proteins whose denatured ensembles behave as those of SN proteins.     

Elementary [Ca2+]i signals generated by electroporation functionally mimic those evoked by hormonal stimulation

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.     

GC base sequence recognition by oligo(imidazolecarboxamide) and C-terminus-modified analogues of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron(II) footprinting studies

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of aromatic transmembrane residues of the delta-opioid receptor in ligand recognition

In the present study we examine the role of transmembrane aromatic residues of the delta-opioid receptor in ligand recognition. Three-dimensional computer modeling of the receptor allowed to identify an aromatic pocket within the helices bundle which spans transmembrane domains (Tms) III to VII and consists of tyrosine, phenylalanine, and tryptophan residues. Their contribution to opioid binding was assessed by single amino acid replacement: Y129F and Y129A (Tm III), W173A (Tm IV), F218A and F222A (Tm V), W274A (Tm VI), and Y308F (Tm VII). Scatchard analysis shows that mutant receptors, transfected into COS cells, are expressed at levels comparable with that of the wild-type receptor. Binding properties of a set of representative opioids were examined. Mutations at position 129 most dramatically affected the binding of all tested ligands (up to 430-fold decrease of deltorphin II binding at Y129A), with distinct implication of the hydroxyl group and the aromatic ring, depending on the ligand under study. Affinity of most ligands was also reduced at Y308F mutant (up to 10-fold). Tryptophan residues seemed implicated in the recognition of specific ligand classes, with reduced binding for endogenous peptides at W173A mutant (up to 40-fold) and for nonselective alkaloids at W274A mutant (up to 65-fold). Phenylalanine residues in Tm V appeared poorly involved in opioid binding as compared with other aromatic amino acids examined. Generally, the binding of highly selective delta ligands (TIPPpsi, naltrindole, and BW373U86) was weakly modified by these mutations. Noticeably, TIPPpsi binding was enhanced at W274A receptor by 5-fold. Conclusions from our study are: (i) aromatic amino acid residues identified by the model contribute to ligand recognition, with a preponderant role of Y129; (ii) these residues, which are conserved across opioid receptor subtypes, may be part of a general opioid binding domain; (iii) each ligand-receptor interaction is unique, as demonstrated by the specific binding pattern observed for each tested opioid compound.     

Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine

The activity of O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from killing by methylating or chloroethylating agents. AGT is strongly inhibited by O6-benzylguanine (ED50, 0.2 microM), and this drug is presently undergoing clinical trials to enhance chemotherapy by alkylating agents. Point mutations such as P140A (ED50, 5 microM) render AGT resistant to O6-benzylguanine (BG). Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG will enable the rational design of improved inhibitors able to inactivate these mutants. BG-resistant AGT mutants may also be valuable for expression in bone marrow stem cells to reduce myelosuppression brought about by alkylating agents, to increase the therapeutic index of therapies including BG, and for use as a selectable marker to allow other genes to be expressed in such stem cells. We have therefore set up a general screen to obtain such mutants by using the ability of AGT to protect Escherichia coli GWR109 lacking endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidine. When the cells were rendered permeable to BG by mutating the lipopolysaccharide membrane component forming strain TRG8, the protection by AGT expression was abolished by treating the cells with BG. The known P140A mutant was used to test the system and was highly selected for by treatment with 50 microM BG and 40 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine. The sequence coding for PVP at positions 138-140 in AGT was replaced with a random nucleotide sequence, and this library was used to transform TRG8. All of the 59 colonies analyzed having AGT activity that survived the selection from the pool of 36,000 transformants were resistant to BG. Many (69%) of these mutants contained lysine at position 140, and all of these showed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated with 1.2 mM BG. This result was confirmed with three mutants (P138K/V139L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogeneity. The next most common residues found at position 140 were arginine (7%) and asparagine (7%). Studies carried out with purified preparations of mutants P140R and P140N revealed that these mutations also provided resistance to BG but to a lesser extent than P140K (ED50s of 190 and 7 microM, respectively). These results indicate that: (a) this screening method can be used to evaluate BG resistance of single or multiple changes throughout the AGT sequence; and (b) replacement of proline-140 with lysine is the most effective point mutation at this site causing BG resistance and is more than 200 times more effective than replacement with alanine.     

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design, synthesis, derivatization, and structure-activity relationships of simplified, tricyclic, 1,2,4-trioxane alcohol analogues of the antimalarial artemisinin

Novel C4-(hydroxyalkyl)trioxanes 5d and 5e were designed and synthesized based on an understanding of the molecular mechanism of action of similar 1,2,4-trioxanes structurally related to the antimalarial natural product artemisinin (1). In vitro efficacies of these two new pairs of C4-diastereomers against chloroquine-sensitive Plasmodium falciparum support conclusions about the importance to antimalarial activity of formation of a C4 radical by a 1,5-hydrogen atom abstraction. Derivatives 6, 7, and 21 of C4 beta-substituted trioxane alcohols 4a, 5d, and 5e were prepared, each in a single-step, high-yielding transformation. Four of these new analogues, 6a-c and 7, are potent in vitro antimalarials, having 140 to 50% of the efficacy of the natural trioxane artemisinin (1).     

Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent manner

The protection against micrococcal nuclease digestion afforded to chromatosomal DNA by the presence of a linker histone (H1(o)) has been quantitatively measured in two reconstituted systems. We have used chromatosomes reconstituted at two distinct positions on a DNA fragment containing the 5S rRNA gene from Lytechinus variegatus and at a specific position on a sequence containing Gal4- and USF-binding sites. In all cases, we find asymmetric protection, with approximately 20 bp protected on one side of the core particle and no protection on the other. We demonstrated through crosslinking experiments that the result is not due to any sliding of the histone core caused by either linker histone addition or micrococcal nuclease cleavage. Because the core particle is itself a symmetric object, the preferred asymmetric location of a linker histone must be dictated by unknown elements in the DNA sequence.