The nucleotide sequence of poliovirus type 3 leon 12 a1b: comparison with poliovirus type 1.

The complete nucleotide sequence of the genome of the Sabin vaccine strain of poliovirus type 3 (P3/Leon 12 a1 b) has been determined from cDNA cloned in E. coli. The genome comprises a 5' non-coding region of 742 nucleotides, a large open reading frame of 6618 nucleotides (89% of the sequence) and a 3' non-coding region of 72 nucleotides. There is 77.4% base-sequence homology and 89.6% predicted amino-acid homology between types 1 and 3. Conservation of all glutamine-glycine and tyrosine-glycine cleavage sites suggests a mechanism of polyprotein processing similar to that established for poliovirus type 1.     

Theory of electrophoresis of reacting systems as applied to the sulfhydryl oxidation of creatine kinase

In an attempt to explain the unusual electrophoretic behavior of fish muscle creatine kinase, a phenomenological theory of transport of reacting systems has been formulated for the electrophoresis of a sulfhydryl protein undergoing oxidation in the presence of a gradient of molecular oxygen. The model assumes slow O2-oxidation of sulfhydryl groups followed by rather rapidly reversible sulfhydryl-disulfide interchange with concomitant change in the electrophoretic mobility of the protein. The computed electrophoretic patterns for this model exhibit a sharp, unimodal ascending boundary but a bimodal descending boundary in which the proportions of the two peaks are time dependent. There is a striking similarity between the theoretical patterns and their experimental counterparts (M.D. Doherty, D.A. Bergman, V.M. Re-Miller, and D.J. Winzor, 1980, Arch. Biochem. Biophys. 202, 558-564); and hence support for consideration of the electrophoresis of fish muscle creatine kinase in these terms.     

Spectroscopic Evidence for Complexing of Acetic Acid with Bovine Serum Albumin, Gramicidin, and Dimethylformamide

Acetic acid has a major effect on the absorption spectra of bovine serum albumin, gramicidin, and dimethylformamide in the region, 255 to 200 mμ. Increasing the concentration of acetic acid causes progressively decreasing absorbency accompanied by a large and progressively increasing red shift of the absorption maximum. The decrease in absorbency is interpreted in terms of a reversible complexing of acetic acid with these molecules and the red shift in terms of a non-specific solvent effect.     

Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages.

Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.     

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.     

Numerical and experimental demonstrations of the need for caution in the use of zonal gel chromatography for characterizing ligand interactions with small acceptors

Quantitative expressions are presented for the evaluation of equilibrium constants for interactions of the type A + B in equilibrium C from experiments entailing the application of a small zone of acceptor-ligand mixture to a column of gel preequilibrated with ligand solution [J.P. Hummel and W.J. Dreyer (1962) Biochim. Biophys. Acta 63, 530-532]. Only in the event that identical elution volumes pertain to acceptor and complex does the steady-state binding constant (Kss) obtained by that method equal the thermodynamic equilibrium constant (K). Simulated elution profiles are then generated with parameters relevant to gel chromatography of the ATP-Mg2+ system on Sephadex G-10 in order to demonstrate the practical importance of the need for distinction between Kss and K in situations where acceptor and complex do not comigrate. A study of the interaction between soybean trypsin inhibitor and cytochrome c by gel chromatography on Sephadex G-75 is then used to illustrate the feasibility of combining information from Hummel-Dreyer experiments with the theoretical expressions to characterize systems under the more general conditions that the elution volumes of A and C differ. A finding of considerable theoretical interest in relation to the simulation of mass migration behavior is the demonstration that a truncation error is the source of zonal spreading in the theoretical-plate model of chromatography. This truncation error is shown to be the source of spreading generated whenever solution of an abbreviated (diffusion-free) continuity equation involves substituting first differences for first derivatives in the differential equation describing mass transport.     

Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit

The large RNA molecule within each ribosomal subunit is folded in a specific and compact form. The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides. The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay. The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state [as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message]. Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state. Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation. Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency. Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state. With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography). We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions.     

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.