[The beta-helical domain of bacteriophage T4 controls the folding of the fragment of long tail fibers in a chimeric protein].

The key stage of the infection of the Escherichia coli cell with bacteriophage T4, the binding to the surface of the host cell, is determined by the specificity of the long tail fiber proteins of the phage, in particular, gp37. The assembly and oligomerization of this protein under natural conditions requires the participation of at least two additional protein factors, gp57A and gp38, which strongly hinders the production of the recombinant form of gp37. To overcome this problem, a modern protein engineering strategy was used, which involves the construction of a chimeric protein containing a carrier protein that drives the correct folding of the target protein. For this purpose, the trimeric beta-helical domain of another protein of phage T4, gp5, was used. It was shown that this domain, represented as a rigid trimeric polypeptide prism, has properties favorable for use as a protein carrier. A fragment of protein gp37 containing five pentapeptides repeats, Gly-X-His-X-His, which determine the binding to the receptors on the bacterial cell surface, was fused in a continuous reading frame to the C-terminus of the domain of gp5. The resulting chimeric protein forms a trimer that has the native conformation of gp37 and exhibits biological activity.

Self-Assembly of Tail Tube Protein of Bacteriophage vB_EcoS_NBD2 into Extremely Long Polytubes in E. coli and S. cerevisiae.

Nucleotides, peptides and proteins serve as a scaffold material for self-assembling nanostructures. In this study, the production of siphovirus vB_EcoS_NBD2 (NBD2) recombinant tail tube protein gp39 reached approximately 33% and 27% of the total cell protein level in Escherichia coli and Saccharomyces cerevisiae expression systems, respectively. A simple purification protocol allowed us to produce a recombinant gp39 protein with 85%⁻90% purity. The yield of gp39 was 2.9 ± 0.36 mg/g of wet E. coli cells and 0.85 ± 0.33 mg/g for S. cerevisiae cells. The recombinant gp39 self-assembled into well-ordered tubular structures (polytubes) in vivo in the absence of other phage proteins. The diameter of these structures was the same as the diameter of the tail of phage NBD2 (~12 nm). The length of these structures varied from 0.1 µm to >3.95 µm, which is 23-fold the normal NBD2 tail length. Stability analysis demonstrated that the polytubes could withstand various chemical and physical conditions. These polytubes show the potential to be used as a nanomaterial in various fields of science.

Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts.

We used a cell-free transcription/translation system to synthesize structural proteins of the T4 bacteriophage. We focused on two proteins that participate in the formation of the virus tail tube assembly. Synthesized separately, the proteins assembled into their in vivo forms, namely one polymerized into rigid hollow nanotubes approximately 20 nm thick and hundreds of nanometers long, the other assembled into 10 nm tube-capping hexameric rings. Co-synthesis of the two proteins, however, revealed a novel structure of a nanodoughnut with an outer diameter of approximately 50 nm and thickness of approximately 20 nm. Cell-free co-synthesis and assembly of T4 structural proteins can be extended in a combinatorial fashion. The addition of other structural genes offers control of native nanoassemblies and may reveal ones not observable by mixing purified components.

A newly synthesized, ribosome-bound polypeptide chain adopts conformations dissimilar from early in vitro refolding intermediates.

Little is known about the conformations of newly synthesized polypeptide chains as they emerge from the large ribosomal subunit, or how these conformations compare with those populated immediately after dilution of polypeptide chains out of denaturant in vitro. Both in vivo and in vitro, partially folded intermediates of the tailspike protein from Salmonella typhimurium phage P22 can be trapped in the cold. A subset of monoclonal antibodies raised against tailspike recognize partially folded intermediates, whereas other antibodies recognize only later intermediates and/or the native state. We have used a pair of monoclonal antibodies to probe the conformational features of full-length, newly synthesized tailspike chains recovered on ribosomes from phage-infected cells. The antibody that recognizes early intermediates in vitro also recognizes the ribosome-bound intermediates. Surprisingly, the antibody that did not recognize early in vitro intermediates did recognize ribosome-bound tailspike chains translated in vivo. Thus, the newly synthesized, ribosome-bound tailspike chains display structured epitopes not detected upon dilution of tailspike chains from denaturant. As opposed to the random ensemble first populated when polypeptide chains are diluted out of denaturant, folding in vivo from the ribosome may begin with polypeptide conformations already directed toward the productive folding and assembly pathway.

Characterization of the protrimer intermediate in the folding pathway of the interdigitated beta-helix tailspike protein.

P22 tailspike is a homotrimeric, thermostable adhesin that recognizes the O-antigen lipopolysaccharide of Salmonella typhimurium. The 70 kDa subunits include long beta-helix domains. After residue 540, the polypeptide chains change their path and wrap around one another, with extensive interchain contacts. Formation of this interdigitated domain intimately couples the chain folding and assembly mechanisms. The earliest detectable trimeric intermediate in the tailspike folding and assembly pathway is the protrimer, suspected to be a precursor of the native trimer structure. We have directly analyzed the kinetics of in vitro protrimer formation and disappearance for wild type and mutant tailspike proteins. The results confirm that the protrimer intermediate is an on-pathway intermediate for tailspike folding. Protrimer was originally resolved during tailspike folding because its migration through nondenaturing polyacrylamide gels was significantly retarded with respect to the migration of the native tailspike trimer. By comparing protein mobility versus acrylamide concentration, we find that the retarded mobility of the protrimer is due exclusively to a larger overall size than the native trimer, rather than an altered net surface charge. Experiments with mutant tailspike proteins indicate that the conformation difference between protrimer and native tailspike trimer is localized toward the C-termini of the tailspike polypeptide chains. These results suggest that the transformation of the protrimer to the native tailspike trimer represents the C-terminal interdigitation of the three polypeptide chains. This late step may confer the detergent-resistance, protease-resistance, and thermostability of the native trimer.

Connection between gene dosage and protein stability revealed by a high-yield production of recombinant proteins in an E. coli LexA1(Ind-) background.

Bacterial production of a plasmid-encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA- LexA+ and RecA+ LexA1(Ind-) backgrounds. In a LexA1(Ind-) context, we have observed lesser toxicity and higher productivity than in the wild-type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression. Also, a negative effect of the incubation temperature on the growth of producing cells was also detected. By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein. Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition. Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth.