Solvent effects on the energy landscapes and folding kinetics of polyalanine

The effect of a solvation on the thermodynamics and kinetics of polyalanine (Ala12) is explored on the basis of its energy landscapes in vacuum and in an aqueous solution. Both energy landscapes are characterized by two basins, one associated with α-helical structures and the other with coil and β-structures of the peptide. In both environments, the basin that corresponds to the α-helical structure is considerably narrower than the basin corresponding to the β-state, reflecting their different contributions to the entropy of the peptide. In vacuum, the α-helical state of Ala12 constitutes the native state, in agreement with common helical propensity scales, whereas in the aqueous medium, the α-helical state is destabilized, and the β-state becomes the native state. Thus solvation has a dramatic effect on the energy landscape of this peptide, resulting in an inverted stability of the two states. Different folding and unfolding time scales for Ala12 in hydrophilic and hydrophobic chemical environments are caused by the higher entropy of the native state in water relative to vacuum. The concept of a helical propensity has to be extended to incorporate environmental solvent effects.

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.

Adenoviral vector-mediated gene transfer offers significant potential for gene therapy of many human diseases. However, progress has been slowed by several limitations. First, the insert capacity of currently available adenoviral vectors is limited to 8 kb of foreign DNA. Second, the expression of viral proteins in infected cells is believed to trigger a cellular immune response that results in inflammation and in only transient expression of the transferred gene. We report the development of a new adenoviral vector that has all viral coding sequences removed. Thus, large inserts are accommodated and expression of all viral proteins is eliminated. The first application of this vector system carries a dual expression cassette comprising 28.2 kb of nonviral DNA that includes the full-length murine dystrophin cDNA under control of a large muscle-specific promoter and a lacZ reporter construct. Using this vector, we demonstrate independent expression of both genes in primary mdx (dystrophin-deficient) muscle cells.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.

A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis. To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas. Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants. Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover. With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg. Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant. Thin-layer and radio-gas chromatographic analyses of the lipids derived from [1-14C]propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides. Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA. The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.

DNA sequences of Alu elements indicate a recent replacement of the human autosomal genetic complement.

DNA sequences of neutral nuclear autosomal loci, compared across diverse human populations, provide a previously untapped perspective into the mode and tempo of the emergence of modern humans and a critical comparison with published clonally inherited mitochondrial DNA and Y chromosome measurements of human diversity. We obtained over 55 kilobases of sequence from three autosomal loci encompassing Alu repeats for representatives of diverse human populations as well as orthologous sequences for other hominoid species at one of these loci. Nucleotide diversity was exceedingly low. Most individuals and populations were identical. Only a single nucleotide difference distinguished presumed ancestral alleles from descendants. These results differ from those expected if alleles from divergent archaic populations were maintained through multiregional continuity. The observed virtual lack of sequence polymorphism is the signature of a recent single origin for modern humans, with general replacement of archaic populations.

Recruitment and replacement of hippocampal neurons in young and adult chickadees: an addition to the theory of hippocampal learning.

We used [3H]thymidine to document the birth of neurons and their recruitment into the hippocampal complex (HC) of juvenile (4.5 months old) and adult blackcapped chickadees (Parus atricapillus) living in their natural surroundings. Birds received a single dose of [3H]thymidine in August and were recaptured and killed 6 weeks later, in early October. All brains were stained with Cresyl violet, a Nissl stain. The boundaries of the HC were defined by reference to the ventricular wall, the brain surface, or differences in neuronal packing density. The HC of juveniles was as large as or larger than that of adults and packing density of HC neurons was 31% higher in juveniles than in adults. Almost all of the 3H-labeled HC neurons were found in a 350-m-wide layer of tissue adjacent to the lateral ventricle. Within this layer the fraction of 3H-labeled neurons was 50% higher in juveniles than in adults. We conclude that the HC of juvenile chickadees recruits more neurons and has more neurons than that of adults. We speculate that juveniles encounter greater environmental novelty than adults and that the greater number of HC neurons found in juveniles allows them to learn more than adults. At a more general level, we suggest that (i) long-term learning alters HC neurons irreversibly; (ii) sustained hippocampal learning requires the periodic replacement of HC neurons; (iii) memories coded by hippocampal neurons are transferred elsewhere before the neurons are replaced.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.

Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.

Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues.

The integrase protein of human immunodeficiency virus type 1 is necessary for the stable integration of the viral genome into host DNA. Integrase catalyzes the 3' processing of the linear viral DNA and the subsequent DNA strand transfer reaction that inserts the viral DNA ends into host DNA. Although full-length integrase is required for 3' processing and DNA strand transfer activities in vitro, the central core domain of integrase is sufficient to catalyze an apparent reversal of the DNA strand transfer reaction, termed disintegration. This catalytic core domain, as well as the full-length integrase, has been refractory to structural studies by x-ray crystallography or NMR because of its low solubility and propensity to aggregate. In an attempt to improve protein solubility, we used site-directed mutagenesis to replace hydrophobic residues within the core domain with either alanine or lysine. The single substitution of lysine for phenylalanine at position 185 resulted in a core domain that was highly soluble, monodisperse in solution, and retained catalytic activity. This amino acid change has enabled the catalytic domain of integrase to be crystallized and the structure has been solved to 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266, 1981-1986]. Systematic replacement of hydrophobic residues may be a useful strategy to improve the solubility of other proteins to facilitate structural and biochemical studies.