Binding of an antiviral agent to a sensitive and a resistant human rhinovirus. Computer simulation studies with sampling of amino acid side-chain conformation. I. Mapping the rotamers of residue 188 of viral protein 1.

The mutation of valine 188 to leucine in the viral protein 1 of human rhinovirus 14 renders the virus resistant to certain antiviral compounds. Thermodynamic-cycle perturbation theory provides a means of calculating the difference in the binding free energies of an antiviral compound to the wild-type virus and to the mutant virus. In calculating the relevant free-energy differences in molecular dynamics simulations, it is important to sample the multiple rotational isomers of residue 188 correctly. In general, these rotamers will not be fully sampled during a single molecular dynamics simulation. However, the contributions of all the rotamers to the free-energy differences associated with mutation of residue 188 may be considered explicitly once they have been identified and their relative free energies determined. Therefore, we describe here the mapping of the rotamers of residue 188 by steric-bump search and energy minimization techniques, and by the computation of potentials of mean force (p.m.f.s.) using umbrella sampling. The usefulness, validity and efficiency of these methods of examining rotameric states is discussed. Adiabatic mapping by energy minimization was found to be unreliable for this residue due to the small magnitude of its interactions with the surrounding protein atoms. Ambiguities in the adiabatic maps were resolved by computing p.m.f.s. The p.m.f. for valine 188 in the unliganded wild-type virus shows a minimum corresponding to the crystallographically observed conformation of valine 188. The p.m.f.s. for valine 188 in the liganded virus and for leucine 188 in the unliganded mutant virus suggest that the experimentally observed conformations may be interpreted as averages of a number of conformations corresponding to those at the minima in the p.m.f.s. The calculations suggest also that the conformation of leucine 188 may change when the ligand binds. The use of the calculated p.m.f.s. to compute the difference in the free energy of binding of an antiviral compound to the wild-type and mutant rhinoviruses is described in the accompanying article.     

Fluorometric microbore amino acid analyzer: the construction of an inexpensive, highly sensitive instrument using o-phthalaldehyde as a detection agent.

The construction of a highly sensitive microbore amino acid analyzer is described. It is based on a standard column chromatographic separation technique with fluorometric detection utilizing o-phthalaldehyde. This analyzer has several desirable features: low column chromatographic pressure, high sensitivity, and easy maintenance. Good precision at a level of 10 pmol is obtained and as little as 0.2 μg protein has been hydrolyzed for composition analysis. It incorporates the use of the single-column method and constant molarity buffers to shorten the analysis time. It is fully automatic and capable of analyzing 130 samples within 7 days with attention required only for reloading 20 samples while the instrument is in operation. Amino acid composition determination based on the peak area and the peak height is discussed.     

Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains. Binding properties of fragments of a conserved eukaryotic RNA binding motif.

We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein.     

Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methionine-containing overlap peptides were isolated. The required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CB1-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 35S, [2,5-3H]histidine, and [5-3H]histidine-labeled Neurospora tyrosinase.     

Elucidation of an A and L system for amino acid transport in the human lymphoblast using a membrane filtration technique.

Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find alpha-AIB to be a poor competitor of alanine and phenylalanine uptake.     

Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt(II) as a spectroscopic probe.

Peptides corresponding to Cys2His2 zinc finger domains from which one amino acid has been deleted have been synthesized and their metal-binding properties characterized. In contrast to earlier reports (Párraga, G., S. Horvath, L. Hood, E. T. Young, and R. E. Klevit. 1990. Proc. Natl. Acad. Sci. USA. 87:137-141.), such peptides do bind metal ions such as cobalt(II). A peptide with the sequence ProTyrLysCysProGluCysLysSerPheSerGlnLysSerAspLeuValLysHisGlnArgThrHis ThrGly (which corresponds to a previously characterized consensus zinc finger sequence from which a Gly residue immediately following the second Cys residue has been deleted) was found to form a 1:1 peptide to cobalt(II) complex with an absorption spectrum quite similar to those previously observed for zinc finger peptide-cobalt(II) complexes. The dissociation constant for this complex is 6 x 10(-6)M, a factor of 100 times higher than that for the parent peptide. A peptide with the sequence LysProTyrProCysGlyLeuCysArgCysPheThrArgArgAspLeuLeulleArgHisAlaGln - LyslleHisSerGlyAsnLeu corresponding to a similar mutation of the peptide ADR1 was also characterized. Spectroscopic studies with cobalt(II) revealed that this peptide forms both 1:1 and 2:1 peptide to cobalt(II) complexes. The absorption spectra of the two forms and the dissociation constants were determined via deconvolution methods. In contrast, the parent peptide ADR1a was found to form only a 1:1 complex under comparable conditions and this 1:1 complex was found to be more stable than that for the mutant. These results reveal that deletion mutations do adversely affect the stability of zinc finger peptide-metal complexes but that the effects are not as drastic as had been previously described.     

Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.     

Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II

Catalysis of the hydration of CO2 by human carbonic anhydrase isozyme II (HCA II) is sustained at a maximal catalytic turnover of 1 mus-1 by proton transfer between a zinc-bound solvent and bulk solution. This mechanism of proton transfer is facilitated via the side chain of His64, which is located 7.5 A from the zinc, and mediated via intervening water molecules in the active-site cavity. Three hydrophilic residues that have previously been shown to contribute to the stabilization of these intervening waters were replaced with hydrophobic residues (Y7F, N62L, and N67L) to determine their effects on proton transfer. The structures of all three mutants were determined by X-ray crystallography, with crystals equilibrated from pH 6.0 to 10.0. A range of changes were observed in the ordered solvent and the conformation of the side chain of His64. Correlating these structural variants with kinetic studies suggests that the very efficient proton transfer (approximately 7 micros-1) observed for Y7F HCA II in the dehydration direction, compared with the wild type and other mutants of this study, is due to a combination of three features. First, in this mutant, the side chain of His64 showed an appreciable inward orientation pointing toward the active-site zinc. Second, in the structure of Y7F HCA II, there is an unbranched chain of hydrogen-bonded waters linking the proton donor His64 and acceptor zinc-bound hydroxide. Finally, the difference in pKa of the donor and acceptor appears favorable for proton transfer. The data suggest roles for residues 7, 62, and 67 in fine-tuning the properties of His64 for optimal proton transfer in catalysis.     

Gene relA function in the expression of amino acid operons. II. Effect of the allelic state of gene relA on the overproduction of threonine by an Escherichia coli K-12 mutant resistant to beta-hydroxynorvaline]

Mutants, resistant to threonine analogue, DL-alpha-amino-beta-hydroxyvaleric acid, were obtained after the treatment of Escherichia coli K-12 RelA- cells with nitrosoguanidine, and among them the strain with maximal threonine production (about 3g/l) was selected. Genetic and biochemical analysis of the producer has revealed the dependency of the threonine production on at least three mutations. The mutation in the thrA gene disturbs retroinhibition of homoserine dehydrogenase by threonine. The mutation in the ilvA gene decreases the activity of threonine deaminase, and thus results in partial isoleucine auxotrophy, and finally, the reversion in the relA gene restores the stringent amino acid control of RNA synthesis in threonine producer cells. The role of relA gene in threonine production was demonstrated by comparing pairs of strains differing from one another in the allelic state of the relA gene. The level of threonine synthesis (its intra- and extracellular concentrations) during moderate isoleucine starvation in RelA+ cells 2-3 times as high as in RelA- cells. The presence of relA+ allele is found to result in the increase of the cell resistance to DL-alpha-amino-beta-hydroxyvaleric acid.     

Primary structure of a human IgA1 immunoglobulin. II. Isolation, composition, and amino acid sequence of the tryptic peptides of the whole alpha1 chain and its cyanogen bromide fragments.

As part of the strategy for determining the covalent structure of a human IgA1 molecule (Bur), a tryptic digest was prepared of the reduced and carboxymethylated alpha1 heavy chain. In addition to the main experiment, tryptic peptides were prepared from the succinylated aminoethylated alpha1 chain and from fragments obtained by CNBr scission of the alpha1 chain. Complete recovery of the peptides was impeded by the large size of some of the tryptic peptides and of the principal CNBr fragment, and difficulty in separating other glycopeptides. Twenty-eight tryptic peptides of the reduced and carboxymethylated alpha1 chain were purified and sequenced, accounting for more than 300 residues. Additional information was obtained by sequence analysis of trypudies described in this series of papers contributed to the complete sequence analysis of the alpha1 chain.     

Complete amino acid sequence of the human alpha 5 (IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient.

We have generated and characterized cDNA clones providing the complete amino acid sequence of the human type IV collagen chain whose gene has been shown to be mutated in X chromosome-linked Alport syndrome. The entire translation product has 1,685 amino acid residues. There is a 26-residue signal peptide, a 1,430-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a Gly-Xaa-Yaa-repeat sequence interrupted at 22 locations, and a 229-residue carboxyl-terminal noncollagenous domain. The calculated molecular weight of the mature alpha 5(IV) chain is 158,303. Analysis of genomic DNA from members of a kindred with Alport syndrome revealed a new HindIII cleavage site within the coding sequence of one of the cDNA clones characterized. The proband had a new 1.25-kilobase HindIII fragment and a lack of a 1.35-kilobase fragment, and his mildly affected female cousin had both alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin. The results were confirmed by allele-specific hybridization analyses.