An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY

The SecY/Sec61α family of membrane proteins are the central subunits of the putative protein translocation channel. We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity. Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function. These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue.

Advances in random matrix theory, zeta functions, and sphere packing

Over four hundred years ago, Sir Walter Raleigh asked his mathematical assistant to find formulas for the number of cannonballs in regularly stacked piles. These investigations aroused the curiosity of the astronomer Johannes Kepler and led to a problem that has gone centuries without a solution: why is the familiar cannonball stack the most efficient arrangement possible? Here we discuss the solution that Hales found in 1998. Almost every part of the 282-page proof relies on long computer verifications. Random matrix theory was developed by physicists to describe the spectra of complex nuclei. In particular, the statistical fluctuations of the eigenvalues (“the energy levels”) follow certain universal laws based on symmetry types. We describe these and then discuss the remarkable appearance of these laws for zeros of the Riemann zeta function (which is the generating function for prime numbers and is the last special function from the last century that is not understood today.) Explaining this phenomenon is a central problem. These topics are distinct, so we present them separately with their own introductory remarks.

Molecular mechanisms of sound amplification in the mammalian cochlea

Mammalian hearing depends on the enhanced mechanical properties of the basilar membrane within the cochlear duct. The enhancement arises through the action of outer hair cells that act like force generators within the organ of Corti. Simple considerations show that underlying mechanism of somatic motility depends on local area changes within the lateral membrane of the cell. The molecular basis for this phenomenon is a dense array of particles that are inserted into the basolateral membrane and that are capable of sensing membrane potential field. We show here that outer hair cells selectively take up fructose, at rates high enough to suggest that a sugar transporter may be part of the motor complex. The relation of these findings to a recent candidate for the molecular motor is also discussed.

Putting ion channels to work: Mechanoelectrical transduction, adaptation, and amplification by hair cells

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer's adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.

Simple neural networks for the amplification and utilization of small changes in neuron firing rates

I describe physiologically plausible “voter-coincidence” neural networks such that secondary “coincidence” neurons fire on the simultaneous receipt of sufficiently large sets of input pulses from primary sets of neurons. The networks operate such that the firing rate of the secondary, output neurons increases (or decreases) sharply when the mean firing rate of primary neurons increases (or decreases) to a much smaller degree. In certain sensory systems, signals that are generally smaller than the noise levels of individual primary detectors, are manifest in very small increases in the firing rates of sets of afferent neurons. For such systems, this kind of network can act to generate relatively large changes in the firing rate of secondary “coincidence” neurons. These differential amplification systems can be cascaded to generate sharp, “yes–no” spike signals that can direct behavioral responses.

Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase.

Recent studies on proteins whose N and C termini are in close proximity have demonstrated that folding of polypeptide chains and assembly of oligomers can be accomplished with circularly permuted chains. As yet no methodical study has been conducted to determine how extensively new termini can be introduced and where such termini cannot be tolerated. We have devised a procedure to generate random circular permutations of the catalytic chains of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and to select clones that produce active or stable holoenzyme containing permuted chains. A tandem gene construct was made, based on the desired linkage between amino acid residues in the C- and N-terminal regions of the polypeptide chain, and this DNA was treated with a suitable restriction enzyme to yield a fragment containing the rearranged coding sequence for the chain. Circularization achieved with DNA ligase, followed by linearization at random with DNase I, and incorporation of the linearized, repaired, blunt-ended, rearranged genes into a suitable plasmid permitted the expression of randomly permuted polypeptide chains. The plasmid with appropriate stop codons also contained pyrI, the gene encoding the regulatory chain of ATCase. Colonies expressing detectable amounts of ATCase-like molecules containing permuted catalytic chains were identified by an immunoblot technique or by their ability to grow in the absence of pyrimidines in the growth medium. Sequencing of positive clones revealed a variety of novel circular permutations. Some had N and C termini within helices of the wild-type enzyme as well as deletions and insertions. Permutations were concentrated in the C-terminal domain and only few were detected in the N-terminal domain. The technique, which is adaptable generally to proteins whose N and C termini are near each other, can be of value in relating in vivo folding of nascent, growing polypeptide chains to in vitro renaturation of complete chains and determining the role of protein sequence in folding kinetics.

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure.

Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.

Novel human DNA alkyltransferases obtained by random substitution and genetic selection in bacteria.

DNA repair alkyltransferases protect organisms against the cytotoxic, mutagenic, and carcinogenic effects of alkylating agents by transferring alkyl adducts from DNA to an active cysteine on the protein, thereby restoring the native DNA structure. We used random sequence substitutions to gain structure-function information about the human O6-methylguanine-DNA methyltransferase (EC 2.1.1.63), as well as to create active mutants. Twelve codons surrounding but not including the active cysteine were replaced by a random nucleotide sequence, and the resulting random library was selected for the ability to provide alkyltransferase-deficient Escherichia coli with resistance to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Few amino acid changes were tolerated in this evolutionarily conserved region of the protein. One mutation, a valine to phenylalanine change at codon 139 (V139F), was found in 70% of the selected mutants; in fact, this mutant was selected much more frequently than the wild type. V139F provided alkyltransferase-deficient bacteria with greater protection than the wild-type protein against both the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine, increasing the D37 over 4-fold and reducing the mutagenesis rate 2.7-5.5-fold. This mutant human alkyltransferase, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of alkylation-based chemotherapeutic regimens.

Active barnase variants with completely random hydrophobic cores.

The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core. If some measure of exquisite, native-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes, either by design or by natural or experimental evolution. To test the minimum requirements for a core to provide sufficient structural integrity for enzymatic activity, we have produced mutants of the ribonuclease barnase in which 12 of the 13 core residues have together been randomly replaced by hydrophobic alternatives. Using a sensitive biological screen, we find that a strikingly high proportion of these mutants (23%) retain enzymatic activity in vivo. Further substitution at the 13th core position shows that a similar proportion of completely random hydrophobic cores supports enzyme function. Of the active mutants produced, several have no wild-type core residues. These results imply that hydrophobicity is nearly a sufficient criterion for the construction of a functional core and, in conjunction with previous studies, that refinement of a crudely functional core entails more stringent sequence constraints than does the initial attainment of crude core function. Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contributed by the hydrophobic core would greatly reduce the initial hurdle on the evolutionary pathway to novel enzymes. Similarly, experimental development of novel functional proteins might be simplified by limiting core design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.

A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo.

Graft-versus-host disease (GVHD) is a T-cell-mediated disease of transplanted donor T cells recognizing host alloantigens. Data presented in this report show, to our knowledge, for the first time that a synthetic copolymer of the amino acids L-Glu, L-Lys, L-Ala, and L-Tyr (molecular ratio, 1.9:6.0:4.7:1.0; Mr, 6000-8500) [corrected], termed GLAT, with promiscuous binding to multiple major histocompatibility complex class II alleles is capable of preventing lethal GVHD in the B10.D2 --> BALB/c model (both H-2d) across minor histocompatibility barriers. Administration of GLAT over a limited time after transplant significantly reduced the incidence, onset, and severity of disease. GLAT also improved long-term survival from lethal GVHD: 14/25 (56%) of experimental mice survived > 140 days after transplant compared to 2/26 of saline-treated or to 1/10 of hen egg lysozyme-treated control mice (P < 0.01). Long-term survivors were documented to be fully chimeric by PCR analysis of a polymorphic microsatellite region in the interleukin 1beta gene. In vitro, GLAT inhibited the mixed lymphocyte culture in a dose-dependent fashion across a variety of major barriers tested. Furthermore, GLAT inhibited the response of nylon wool-enriched T cells to syngeneic antigen-presenting cells presenting minor histocompatibility antigens. Prepulsing of the antigen-presenting cells with GLAT reduced the proliferative response, suggesting that GLAT inhibits antigen presentation.