Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases.

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.

DNA shuffling of subgenomic sequences of subtilisin.

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.

Directed evolution of a thermostable esterase.

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Combinatorial protein design by in vitro recombination.

DNA recombination is a powerful engine for the creation of new phenotypes. Recently, methods for in vitro DNA recombination (DNA shuffling) have been developed and applied to the evolution of novel molecules in the laboratory. An exciting new development is the shuffling of homologous genes to create diversity for directed evolution.

Molecular evolution by staggered extension process (StEP) in vitro recombination.

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.

Random-priming in vitro recombination: an effective tool for directed evolution.

A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.

Expression of small, therapeutic RNAs in human cell nuclei.

Effective intracellular expression of small RNA therapeutics depends on a number of factors. The RNA, whether antisense, ribozyme, or RNA aptamer, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where it can be most effective. To overcome a number of these problems we have been testing expression cassettes based on the human tRNA(met) and U6 snRNA promoters, in which transcripts encoding small RNA inserts are protected against attack from the 3' and Transient expression in cultured cells results in 10(9)-2 x 10(7) full-length transcripts per cell, depending partially on the promoter construct used but also on the nature of the insert RNA 5' gamma-Phosphate methylation (capping) depended, as expected, on the inclusion of specific U6 snRNA sequences from positions +19 to +27. In situ localization of the transcripts shows that both tRNA and U6 promoter transcripts give primarily punctate nuclear patterns, and that capping of transcripts is not required for nuclear retention. Several different insert RNAs directed against HIV-1 were tested by cotransfection with HIV-1 provirus and assay for subsequent viral reverse transcriptase production. These include antisense RNA, hairpin and hammerhead ribozymes, and RNA ligands (aptamers) for Tat and Rev RNA binding proteins. Results show that Rev-binding RNAs efficiently block HIV-1 gene expression, whereas other RNAs have little or no effected when expressed in these cassettes.

Incorporation of a non-nucleotide bridge into hairpin oligonucleotides capable of high-affinity binding to the Rev protein of HIV-1.

A bridge containing a rigid trans-stilbene group, -P(O)(O-)O(CH2)3NHC(O)- C6H4-CH=CHC6H4C(O)NH(CH2)3OP(O)(O-)-, has been incorporated into several oligonucleotide sequences based on the minimal Rev Binding Element (RBE) of HIV-1. This bridge was found to be effective as a UUCG tetraloop in stabilizing short RNA duplex structures containing mismatched bases and bulged out nucleotide residues and to be more effective than either a TTTT loop or a triethyleneglycol linker in stabilizing similar DNA structures. Evaluation of stilbene-containing RNA RBE sequences of varying length for their ability to bind the Rev protein of HIV-1 showed that a 22-nucleotide stilbenedicarboxamide conjugate bound Rev almost as well as a 94-base fragment of the Rev Responsive Element (RRE). A DNA hairpin mimetic with the same sequence was incapable of Rev binding. Taken together, these experiments serve as an example for how in vitro selection and chemical modification can be combined to generate high-affinity mimetics of nucleic acid sequence and structure.

RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo.

RNA aptamers (binding sequences) that can interact tightly and specifically with the human immunodeficiency virus type 1 Rev protein have previously been selected from random sequence pools. Although the selected sequences compete with the wild-type Rev-binding element (RBE) in vitro, it was not known whether they would be able to functionally replace the RBE in vivo. Two aptamers that were different from the wild-type RBE in terms of both primary sequence and secondary structure were inserted into the full-length Rev-responsive element (RRE) in place of the RBE. The hybrid RREs were assayed for their ability to mediate Rev function in vivo using a reporter system. The aptamers were found to be functionally equivalent to the wild-type element when the assay system was saturated with Rev and better than the wild-type element when Rev was limiting. These results demonstrate that the affinity of the primary Rev-binding element rather than its particular sequence may be most responsible for conferring Rev responsiveness on viral mRNAs. Moreover, the fact that increased binding ability can lead to increased Rev responsiveness suggests that cellular factors do not directly influence the Rev:RBE interaction. Finally, since sequences distinct from the RBE are found to be Rev responsive, it may be possible for the RBE to readily mutate in response to drugs or gene therapy reagents that target the Rev:RBE interaction.

A simple code for protein:RNA interactions.

The Tat and Rev proteins of HIV-1 and the Rex protein of HTLV-I do not interact with their cognate ligands via a particular structural motif but instead specifically recognize RNA molecules by using agglomerations of arginine residues (1). These proteins are members of the so-called arginine-rich motif (ARM) family. There is little data to support (or contradict) the hypothesis that a few simple arginine:RNA interactions govern how ARMs recognize their viral targets. Not only is it unclear how ARM proteins other than Tat interact with their cognate RNA ligands, for the most part it is not even known how structurally complex these RNA ligands are. In order to fully explore the range of RNA sequences and structures that can bind to ARMs we have carried out in vitro genetic selections with two disparate viral proteins: Rev and Rex.

High resolution matrix-assisted laser desorption/ionization time-of-flight analysis of single-stranded DNA of 27 to 68 nucleotides in length.

Mass spectra of single-stranded DNA oligonucleotides were acquired using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Resolution enhancement using space-velocity correlation focusing allows for facile observation of different oligomers, the direct observation of individual DNA-metal adducts, and investigation of counter ion structure.

Metabolic activity of microorganisms in evaporites.

Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative. Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months. In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen. Denitrification was not observed. Our results quantified the slow microbial activity that can occur in salt crystals. Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community.

Selection and design of high-affinity RNA ligands for HIV-1 Rev.

We have used in vitro selection to isolate minimal, high-affinity RNA ligands for the Rev protein of HIV-1. Sequence analysis reveals that the tightest binding aptamers exhibit some similarity to a Rev-binding element (RBE) localized within the Rev-responsive element (RRE), but also contain novel sequence and structural motifs. A short helical stem and bulged nucleotides (nt) CUC ... UYGAG that have no counterpart in the wild-type (wt) element contribute to high-affinity binding. We have designed and synthesized a short (37 nt) RNA molecule that incorporates this motif; this RNA ligand has from three- to fivefold tighter binding than the full-length wt element, and up to 16-fold tighter than minimal wt RBEs. A guanosine:guanosine pairing that is postulated to occur in the wt element has been altered to other base pairings in the context of our optimized minimal element. RNAs that contain non-Watson-Crick base pairings, that can be modeled as isosteric to the wt G:G pair, bind Rev up to 160-fold tighter than elements that contain canonical Watson-Crick pairings or non-isosteric mismatches. These results support the hypothesis that Rev recognizes structural features associated with a non-Watson-Crick base pair.

Selective optimization of the Rev-binding element of HIV-1.

RNA molecules that can bind to the Rev protein of HIV-1 have been isolated from random sequence nucleic acid pools based on a minimal Rev-binding element (RBE) found within the Rev Responsive Element (RRE). While the selected sequences are related to the wild-type element, they also contain substitutions that allow them to bind Rev up to 10-fold better in vitro. A hypothesized homopurine pairing at G48:G71 is generally replaced by A48:A71; the occasional selection of C48:A71 suggests that R71 may be in a syn conformation. These data support the structural model for the RBE originally proposed by Bartel et al. (1). Additional interactions with the Rev protein are promoted by the sequence CUC ... UYGAG, found in one class of high-affinity aptamers, but absent from the wild-type element. Within each class of aptamers different residues and substructures covary with one another to generate optimal Rev-binding surfaces. The interdependencies of different nucleotide substitutions suggest structural models for both the wild-type RBE and the selected high-affinity aptamers.

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.

Triton: do we see to the surface?

The quantity and physical state of methane and nitrogen in the atmosphere of Neptune's satellite Triton and on the surface are evaluated by means of new telescopic data and laboratory measurements of these volatiles. Methane ice is seen in some spectral regions, indicating that the atmosphere is sufficiently transparent to permit sunlight penetration to the surface. Some of the molecular nitrogen absorption occurs in the atmosphere, though some must occur in condensed nitrogen (liquid or solid) on Triton's surface, or in a thin cloud of condensed nitrogen. The Voyager spacecraft cameras should see the surface of Triton.

Production of organic compounds in plasmas: a comparison among electric sparks, laser-induced plasmas, and UV light.

The chemistry in planetary atmospheres that is induced by processes associated with high-temperature plasmas is of broad interest because such processes may explain many of the chemical species observed. There are at least two important phenomena that are known to generate plasmas (and shocks) in planetary atmospheres: lightning and meteor impacts. For both phenomena, rapid heating of atmospheric gases leads to formation of a high-temperature plasma which emits radiation and produces shock waves that propagate through the surrounding atmosphere. These processes initiate chemical reactions that can transform simple gases into more complex compounds. In order to study the production of organic compounds in plasmas (shocks), various mixtures of N2, CH4, and H2, modeling the atmosphere of Titan, were exposed to discrete sparks, laser-induced plasmas (LIP), an ultraviolet radiation. The yields of HCN and several simple hydrocarbons were measured by gas chromatography and compared to those calculated from a simple quenched thermodynamic equilibrium model. The agreement between experiment and theory was fair for HCN and C2H2. However, the agreement for C2H6 and the other hydrocarbons was poor, indicating that a more comprehensive theory is needed. Our experiments suggest that photolysis by ultraviolet light from the plasma is an important process in the synthesis. This was confirmed by the photolysis of gas samples exposed to the light but not to the shock waves emitted by the sparks. Hence, the results of these experiments demonstrate that the thermodynamic equilibrium theory does not adequately model lightning and meteor impacts and that photolysis must be included. Finally, the similarity in yields between the spark and the LIP experiments suggest that LIP provide valid and clean simulations of lightning and meteor impacts and that photolysis must be included. Finally, the similarity in yields between the spark and the LIP experiments suggests that LIP provide valid and clean simulations of lightning in planetary atmospheres.

Lightning production of hydrocarbons and HCN on Titan: laboratory measurements.

Many hydrocarbon species have been detected in the atmosphere of Titan. It is possible that lightning activity is occurring in the troposphere and that it contributes to the hydrocarbon inventory. Measurements of the chemical yields of hydrogen cyanide, acetylene, ethylene, ethane, and propane from simulated lightning discharges are reported. A comparison of the experimental results with those based on thermodynamic equilibrium assumptions shows significant disagreement and implies that theories based solely on thermodynamic equilibrium are inadequate. Although photochemistry and charged particle chemistry occurring in the stratosphere can account for many of the observed hydrocarbon species, the predicted abundance of ethylene is too low by a factor of 10 to 40. While some ethylene will be produced by charged-particle chemistry, the production of ethylene by lightning and its subsequent diffusion into the stratosphere appears to be an adequate source.