Folding kinetics of phage 434 Cro protein

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.     

Folding of the reduced form of the thioredoxin from bacteriophage T4

The folding pattern for bacteriophage T4 thioredoxin is similar to that of the oxidized form [Borden, K. L. B., & Richards, F. M. (1990) Biochemistry 29, 3071-3077]. Equilibrium and kinetic studies were carried out by fluorescence and circular dichroism techniques. The same box model proposed for the oxidized form, with four identifiable states, can accommodate most of the data: N----Uc----Ut----It----N, where N is the native state, Uc is the unfolded species with Pro 66 in the cis form, Ut is the unfolded species with Pro 66 in the trans form, and It is a trans-Pro 66 intermediate with a volume comparable to that of N. However, the relative importance of the different components is shifted between the oxidized and reduced proteins. In spite of the small size of the disulfide loop, the Cys 14-Cys 17 bond appears to be important in stabilizing It. The tertiary structure as monitored by near-UV CD and fluorescence indicates that the reduced form is significantly less stable than its oxidized counterpart; however, the two secondary structures, as seen by far-UV CD, are very similar. The intermediate It behaves as though it is cold denaturated at 4 degrees C.     

Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C.

The kinetics of unfolding and refolding of T4 lysozyme and nine of its mutants have been investigated as a function of guanidinium chloride concentration at 12 degrees C. All show simple two-state, first-order kinetics. Two types of mutants were studied: proline-alanine interchanges and substitutions at position 3 with side chains of varying hydrophobicity. Crystal structures are available for seven of the ten proteins. The effect of mutations on the folding kinetics is more pronounced and complex than on equilibrium thermodynamics. The proteins fall into two broad kinetic classes with one class rather close to the wild type. P86A is a mutant with marked changes in kinetics but only a very small change in stability. Since the 86 position is in the middle of an alpha-helix, the indications are that the helix containing an A residue is more stable in the transition state than one containing a P residue. The other mutants are more complicated, with the refolding and unfolding rates unequally affected by the mutations. On the basis of comparisons with other investigations, we conclude that the rate-determining step in the presence of guanidinium chloride is not the same as in aqueous solution and that it most likely precedes it. The indications are that we are studying the formation of a transition intermediate which is destabilized by the denaturant and which resembles the A intermediate of the framework or molten globule models for protein folding.     

Single turnover kinetics of phage T4 DNA-(N6-adenine)methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase [EC 2.1.1] was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k1 = 0.21 s-1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k2 = 0.023 s-1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI Mtase. Substitutions of bases and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme-substrate complexes is usually sharply reduced. The flipping of the adenosine residue, a target for the modification in the recognition site, revealed by fluorescence titration, upon interaction with the enzyme supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms

Both T4 phage and DNA-free ghosts inhibit replication of RNA phage f2. Most but not all of the effects by T4 upon f2 growth can be blocked by the addition of rifampicin prior to T4 superinfection; by contrast, the inhibition of f2 synthesis by T4 ghosts cannot be blocked by rifampicin. This indicates that inhibition by intact T4 requires gene function, while inhibition by ghosts does not. There is a small, multiplicity-dependent inhibition by viable T4 on f2 growth in the presence of rifampicin which may be similar to the gene function-independent inhibition by T4 ghosts. With one viable T4 per cell, there appears to be no effect by viable T4 upon f2 growth which does not require T4 gene action. Moreover, increasing multiplicities of viable T4 appear to inhibit T4 replication as well.In the absence of rifampicin, pre-existing f2 single and double-stranded RNA are degraded after superinfection by viable T4, but remain stable after superinfection by ghosts. However, no new f2 RNA is synthesized after superinfection with either. In the presence of rifampicin, f2-specific protein synthesis is largely unaffected by viable T4, but is completely inhibited by ghosts. Both Escherichia coli, as well as f2-speciflc polysomes disappear in the presence of ghosts.We conclude that, at low multiplicities, T4 phage and T4 ghosts inhibit replication of f2 phage, and presumably host syntheses, by different mechanisms.     

Radiation sensitive mutants of phage T4

Summary A comparative series of phage survival, multiplicity reactivation and radiation stability experiments have been performed with single and double combinations of the four UV-sensitive mutations v, x, y and 1206. These mutations appear to fall into two groups with v (which is associated with excision repair) in one, and x, y and 1206 (associated with what can be non-committally termed replication repair) in the other. The pattern of the results suggest that the extent of the initial shoulder exhibited by multiplication reactivation curves is determined by replication repair, while the slope of the subsequent linear portion depends on excision repair. The development of the characteristic radiation stability observed in Luria-Latarjet experiments is shown to depend to a major extent on replication repair and only in a minor way on excision repair.     

Folding kinetics of mammalian ribonucleases

The folding kinetics of seven different pancreatic ribonucleases are compared both under native conditions and within the unfolding transition. In general, the folding kinetics of these proteins are similar despite numerous amino acid substitutions. Ribonucleases with 4-6 proline residues show 80% slow-folding species. For three ribonucleases with 7 prolines this number increases to 90%. Porcine ribonuclease with a unique Pro 114-Pro 115 sequence folds significantly slower than other ribonucleases which do not show this sequence.     

Gene function of heterozygotes in phage T4

Summary Gene function of various T4-heterozygotes was tested. About half of the HETs containing wild type and anam-mutation disappeared under non-permissive conditions, if theam-defect concerned early functions. The same was found when phages, heterozygous forr ⁺ and anrII-point-mutation, were adsorbed to K12 (). A much more extensive loss of HETs in K could be observed if anrIIA- and anrIIB-point-mutation (block-mutations showed different results) occurred together in a non-recombinant heterozygote. The findings provide evidence that one class of T4-heterozygotes has a heteroduplex DNA-structure.With 3 Figures in the Text     

The pathway of recombination in phage T4

Summary A study has been made of the effect of modifying the products of the early T4 genes on the frequency with which haploid segregants are generated by recombination from a phage harbouring a standard genetic duplication. Alterations in the products of genes 32, 44, 46, 47 and 59 have been found to significantly decrease the segregation frequency and are, therefore, considered to be involved in the T4 recombination pathway.     

Atomic coordinates for T4 phage lysozyme.

Atomic coordinates are presented for the lysozyme from T4 bacteriophage. The coordinates were derived from a 2.4 Å resolution electron density map based on two isomorphous heavy-atom derivatives, interpreted in terms of the known amino acid sequence, and adjusted to have stereochemically acceptable bond lengths and angles.     

Hybrid transfer RNA genes in phage T4

We describe the isolation and characterization of two unusual amber suppressor forms of T4 tRNALeu. The sequences of the suppressor tRNAs can be described as hybrids of wild-type tRNALeu and suppressor tRNAGln molecules: the chain lengths and majority of the nucleotide residues corresponded to tRNALeu, but CUA anticodons flanked by 2-14 residues were identical to tRNAGln. The uncertainty as to the exact number of flanking residues correlated with tRNAGln is due to the similarity of the two tRNA sequences in this region. No evidence was found for changes in other T4 tRNAs. We propose that genes for the hybrid tRNAs were produced by mispairing of DNAs at anticodon segments of tRNALeu and tRNAGln with a double crossover flanking those segments.     

T4 bacteriophage as a phage display platform

Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems.     

The structure of rII diploids of phage T4

Summary Crosses between the rII deletion 1589 and an overlapping deletion such as 638 which lies entirely within the rIIB cistron generate a few T4 phage particles, the so-called rII diploids, which contain two copies of the rII region, one derived from each parent in the cross. A specific model is proposed to account for the properties of these rII diploids. This model postulates: 1) the rII diploids contain a tandem duplication, 2) the duplicated region extends both to the left and right of the rII region itself, and 3) during phage multiplication recombination occurs between homologous regions of the duplication. These assumptions lead to precise predictions on the following points: 1) the frequency at which haploid 1589 and 638 phage particles are generated during multiplication, 2) the ratio of 1589 and 638 phage amongst the segregants, 3) the relative lengths of terminal redundancy to be found in the rII diploids, the 1589 and 638 segregants and wild-type T4, and 4) the formation and properties of homozygous diploids containing two copies either of the 1589 or 638 region.Experiments are reported which validate the model on these points and also indicate how the homozygous diploids can be utilized to generate new rII diploids with structures which would otherwise be unobtainable.