De novo design, expression, and characterization of Felix: a four-helix bundle protein of native-like sequence.

The protein Felix was designed de novo to fold into an antiparallel four-helix bundle of specific topology. Its sequence of 79 amino acid residues is not homologous to any known protein sequence, but is "native-like" in that it is nonrepetitive and contains 19 of the 20 naturally occurring amino acids. Felix has been expressed from a synthetic gene cloned in Escherichia coli, and the protein has been purified to homogeneity. Physical characterization of the purified protein indicates that Felix (i) is monomeric in solution, (ii) is predominantly alpha-helical, (iii) contains a designed intramolecular disulfide bond linking the first and fourth helices, and (iv) buries its single tryptophan in an apolar environment and probably in close proximity with the disulfide bond. These physical properties rule out several alternative structures and indicate that Felix indeed folds into approximately the designed three-dimensional structure.

Directed mutagenesis of dihydrofolate reductase.

Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.

De novo design of enzyme inhibitors by Monte Carlo ligand generation.

A new computational method for the in situ generation of small molecules within the binding site of a protein is described. The method has been evaluated using two well-studied systems, dihydrofolate reductase and thymidylate synthase. The method has also been used to guide improvements to inhibitors of HIV-1 protease. One such improvement resulted in a compound selected for preclinical studies as an antiviral agent against AIDS.

High-level expression of self-processed HIV-1 protease in Escherichia coli using a synthetic gene.

A synthetic gene coding for HIV-1 protease (PR) has been constructed and a system for its efficient expression in E. coli has been established: PR is synthesized as a fusion protein with E. coli dihydrofolate reductase under the control of a bacteriophage T7 promoter. The synthetic gene was constructed to enable rapid construction of defined mutants by restriction fragment replacement. A set of mutants has been constructed which may facilitate elucidation of the mechanism of PR self-cleavage from polyprotein precursors. We have demonstrated that the C-terminal residue (Phe99 in the native sequence) of the processing intermediate is absolutely required for subsequent cleavage at the N-terminal cleavage site. The potential structural role of this residue is discussed with reference to the recently published HIV-1 PR structure.

tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter.

The yeast SUP53 gene encodes a leucine-inserting amber suppressor tRNA. We have introduced specific base substitutions into both 5' and 3' elements of the intragenic promoter of this tRNA gene. The influence of these sequence changes on promoter function has been investigated by transcription of the mutant genes in a homologous cell-free system. Our results do not support the idea that tertiary intragenic structure is important in tRNA gene transcription. For one of the SUP53 mutants we are able to suggest a plausible molecular basis for defective transcription: a single base substitution in the 3' element of the intragenic promoter prevents the interaction of this element with a putative transcription factor.

Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.

The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented. A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors. The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction. Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease.

Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein.

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.

Changing the identity of a transfer RNA.

A leucine transfer RNA has been transformed into a serine transfer RNA by changing 12 nucleotides. This result indicates that a limited set of residues determine tRNA identity.

Primary structure of the M subunit of the reaction center from Rhodopseudomonas sphaeroides.

The reaction center is a membrane-bound bacteriochlorophyll-protein complex that mediates the primary photochemical events in the photosynthetic bacterium Rhodopseudomonas sphaeroides. The previously determined amino-terminal sequences of the three subunits of the reaction center protein were used to design synthetic mixed oligonucleotide probes for the structural genes encoding the subunits. One of these probes was used to isolate and clone a fragment of DNA from R. sphaeroides that contained the gene encoding the M subunit. The nucleotide sequence of this gene was determined by the dideoxy method. In addition, a number of tryptic and chymotryptic peptides from the M protein were isolated and subjected to sequence analysis, and the sequence of the carboxyl terminus was determined. Together with the amino-terminal sequence, the data establish the primary structure of the M protein. The distribution of hydrophobic residues in the amino acid sequence suggests the presence of five membrane-spanning segments. A significant homology was found between the amino acid sequence of the M subunit and a thylakoid membrane protein (M(r) 32,000) from spinach that has been implicated in herbicide and quinone binding.

Splicing of yeast tRNA precursors: a two-stage reaction.

Soluble extracts of S. cerevisiae splice tRNA precursors which contain intervening sequences. The reaction goes to completion and requires ATP for the production of mature sequence tRNA. In the absence of ATP, half-tRNA molecules accumulate. Similar half-tRNA molecules appear as kinetic intermediates and accumulate if splicing is inhibited with pure, mature tRNA. Half-tRNA molecules have been purified. These half-tRNAs are efficiently ligated in an ATP-dependent reaction that is inhibited by added mature tRNA. The product of ligation is the expected mature sequence tRNA. The excised intervening sequence has also been identified. These results suggest an enzymatic mechanism for splicing which involves two independent steps.

Splicing of yeast tRNA precursors: structure of the reaction intermediates.

The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.

Recombination of the bph (Biphenyl) Catabolic Genes from Plasmid pWW100 and Their Deletion during Growth on Benzoate.

Pseudomonas sp. strain CB406 was isolated from polychlorinated biphenyl-contaminated soil and harbors a nontransmissible plasmid, pWW100, of approximately 200 kb which carries the genes required for biphenyl and 4-chlorobiphenyl catabolism. The catabolic phenotype was mobilized following the construction in vivo of a cointegrate plasmid containing functional upper and lower biphenyl operons inserted into the broad-host-range R plasmid RP4. The Bph phenotype carried by pWW100 was stable in nonselective media but was unstable during growth on benzoate, where the sequential selection of two species of bph deletion derivatives occurs at high frequency. This mirrors observations made with TOL plasmids (encoding toluene and xylene catabolism) grown under similar conditions. Subcloning of dioxygenase genes involved in biphenyl catabolism confirmed the localization of the bph genes on the wild-type plasmid and the RP4 cointegrate plasmid.

In vitro transcription and processing of a yeast tRNA gene containing an intervening sequence.

A gene for Saccharomyces cerevisiae tRNATrp has been sequenced which contains an intervening sequence of 34 bp (H. S. Kang and J. Abelson, unpublished results). The mutant yeast strain ts-136 accumulates a precursor to tRNATrp which contains mature ends and is colinear with the tRNATrp gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNATrp gene contained on plasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence. The Xenopus extract accurately splices the precursor transcript to mature-sized tRNATrp.

Cloning of the Vibrio harveyi luciferase genes: use of a synthetic oligonucleotide probe.

A mixed-sequence synthetic oligonucleotide probe was used to isolate a clone containing the gene encoding the alpha subunit of bacterial luciferase from Vibrio harveyi and part of the gene coding for the beta subunit. DNA sequence analysis has allowed us to determine that the genes are closely linked on the bacterial chromosome and transcribed in the same direction. Comparison of the sequences in the regions preceding the two structural genes has revealed considerable homology and has identified sites that may be involved in the expression of the genes. Identification of a clone from a clone bank of total genomic DNA from this organism shows that mixed probes can be successfully used to isolate a gene of interest from any bacterium provided some protein sequence for the gene product is available.

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component.

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.