Selection for improved protein stability by phage display.

A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display.     

Nucleotide sequence of Bacillus phage Nf terminal protein gene.

The nucleotide sequence of Bacillus phage Nf gene E has been determined. Gene E codes for phage terminal protein which is the primer necessary for the initiation of DNA replication. The deduced amino acid sequence of Nf terminal protein is approximately 66% homologous with the terminal proteins of Bacillus phages PZA and luminal diameter 29, and shows similar hydropathy and secondary structure predictions. A serine which has been identified as the residue which covalently links the protein to the 5' end of the genome in luminal diameter 29, is conserved in all three phages. The hydropathic and secondary structural environment of this serine is similar in these phage terminal proteins and also similar to the linking serine of adenovirus terminal protein.     

Mechanism of phage P22 tailspike protein folding mutations.

Temperature-sensitive folding (tsf) and global-tsf-suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off-pathway aggregation, the kinetics of refolding after dilution from acid-urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants. At low temperature (10 degrees C), subunit-folding rates, measured as an increase in fluorescence, were similar for wild-type and mutants. At 25 degrees C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf-mutant background, but had no effect in the wild-type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off-pathway reaction, as revealed by temperature down-shifts after varied times during refolding at high temperature (40 degrees C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate. In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up-shifts during refolding at 25 degrees C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit-folding intermediates and were also observable during thermal unfolding of the native protein.     

Portal protein diversity and phage ecology

Oceanic phages are critical components of the global ecosystem, where they play a role in microbial mortality and evolution. Our understanding of phage diversity is greatly limited by the lack of useful genetic diversity measures. Previous studies, focusing on myophages that infect the marine cyanobacterium Synechococcus, have used the coliphage T4 portal-protein-encoding homologue, gene 20 (g20), as a diversity marker. These studies revealed 10 sequence clusters, 9 oceanic and 1 freshwater, where only 3 contained cultured representatives. We sequenced g20 from 38 marine myophages isolated using a diversity of Synechococcus and Prochlorococcus hosts to see if any would fall into the clusters that lacked cultured representatives. On the contrary, all fell into the three clusters that already contained sequences from cultured phages. Further, there was no obvious relationship between host of isolation, or host range, and g20 sequence similarity. We next expanded our analyses to all available g20 sequences (769 sequences), which include PCR amplicons from wild uncultured phages, non-PCR amplified sequences identified in the Global Ocean Survey (GOS) metagenomic database, as well as sequences from cultured phages, to evaluate the relationship between g20 sequence clusters and habitat features from which the phage sequences were isolated. Even in this meta-data set, very few sequences fell into the sequence clusters without cultured representatives, suggesting that the latter are very rare, or sequencing artefacts. In contrast, sequences most similar to the culture-containing clusters, the freshwater cluster and two novel clusters, were more highly represented, with one particular culture-containing cluster representing the dominant g20 genotype in the unamplified GOS sequence data. Finally, while some g20 sequences were non-randomly distributed with respect to habitat, there were always numerous exceptions to general patterns, indicating that phage portal proteins are not good predictors of a phage's host or the habitat in which a particular phage may thrive.     

In vitro folding pathway of phage P22 tailspike protein.

The intracellular chain folding and association pathway of the thermostable, trimeric phage P22 tailspike endorhamnosidase has been the subject of a previous detailed study employing temperature-sensitive folding mutants. Recently, reconstitution of native tailspikes from completely unfolded polypeptides has been accomplished, providing a model system to compare protein folding pathways in vivo and in vitro. The in vitro reconstitution pathway of the protein after dilution from guanidine hydrochloride or acid-urea solutions at 10 degrees C was characterized by spectroscopic and hydrodynamic techniques, and may be summarized as an ordered sequence of folding, association, and folding reactions. Multiphasic folding of monomers was indicated by changes in circular dichroism and fluorescence, with a rate constant of k = 1.6 X 10(-3) s-1 for the slowest phase observed spectroscopically. Trimerization of structured monomers was followed by size-exclusion HPLC and was completed within 1.5 h at a protein concentration of 20 micrograms/mL. Although at this time trimers did not exchange subunits, they were readily dissociable by dodecyl sulfate in the cold. Formation of native, detergent-resistant trimers was only completed after 3 days of reconstitution at 10 degrees C. The reconstitution pathway of the tailspike protein closely resembles its intracellular maturation path. Thus, the in vitro reconstitution system, as a valid model of chain folding and association in vivo, should provide the tools to localize the steps or intermediates on the pathway that are the targets of temperature-sensitive folding mutations.     

Filamentous phage infection: required interactions with the TolA protein.

Infection of Escherichia coli by the filamentous phage f1 is initiated by binding of the phage to the tip of the F conjugative pilus via the gene III protein. Subsequent translocation of phage DNA requires the chromosomally encoded TolQ, TolR, and TolA proteins, after the pilus presumably has withdrawn, bringing the phage to the bacterial surface. Of these three proteins, TolA is proposed to span the periplasm, since it contains a long helical domain (domain II), which connects a cytoplasmic membrane anchor domain (domain I) to the carboxyl-terminal domain (domain III). By using a transducing phage, the requirement for TolA in an F+ strain was found to be absolute. The role of TolA domains II and III in the infective process was examined by analyzing the ability of various deletion mutants of tolA to facilitate infection. The C-terminal domain III was shown to be essential, whereas the polyglycine region separating domains I and II could be deleted with no effect. Deletion of helical domain II reduced the efficiency of infection, which could be restored to normal by retaining the C-terminal half of domain II. Soluble domain III, expressed in the periplasm but not in the cytoplasm or in the medium, interfered with infection of a tolA+ strain. The essential interaction of TolA domain III with phage via gene III protein appears to require interaction with a third component, either the pilus tip or a periplasmic entity.     

Lysis protein S of phage lambda functions in Saccharomyces cerevisiae.

The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control. Induction with galactose in S. cerevisiae terminated cell growth and prevented colony formation. Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene. These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.     

Efficiency of phage protein synthesis in lambda-infected E. coli minicells

Phage lambda major head protein, the gene E product, has been identified among other phage proteins synthesized in lambda-infected Escherichia coli minicells, separated by SDS-acrylamide gel electrophoresis. On stained gels, the same protein has also been detected among total (bacterial and phage) proteins of lambda-infected minicells. The contribution of lambda proteins to the total protein content of lambda-infected minicells was found to be about 12% following 30 min lambda-infection. The inhibition of lambda early protein synthesis (shown by other authors in nucleate bacterial cells) practically does not occur in minicells; this may be the reason of the observed high efficiency of phage protein synthesis.     

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.     

Oligomeric structures of the phage phi29 histone-like protein p6.

Protein p6 of Bacillus subtilis phage phi29 has been described as a histone-like protein, playing a role in genome organization and compaction, on the basis of its high intracellular abundance, its pleiotropic effect, and its ability to bind and highly compact the whole phi29 DNA in vitro. Protein p6 forms large multimeric nucleoprotein complexes in which a right-handed superhelical DNA wraps toroidally around the protein core. Analytical ultracentrifugation analysis, at the concentration estimated in vivo (at least 1 mM), showed that protein p6 self-associates into elongated oligomers, suggesting that, in the absence of DNA, the protein could form a scaffold for DNA binding. In this work we have studied the structure of these oligomers by transmission electron microscopy and image processing. The results show that protein p6 aggregates into crooked-shaped oligomers, compatible with a helical structure. The oligomers could interact head-to-tail to form doughnut-shaped structures or they could grow into right-handed double-helical filaments by a nucleation-dependent polymerization process. The dimensions of the crooked-shaped structures are in agreement with that of the DNA in the nucleoprotein complex previously described. We propose that the crooked-shaped structures could act as a scaffold imposing the right-handed path followed by the DNA, and thus it could be considered a non-transient DNA chaperone.     

Mechanism of protein priming DNA replication of B.subtilis phage M2.

B.subtilis phage M2 uses a protein, instead of RNA, as the primer of its DNA replication. Hence this protein encoded in the phage genome is called as the primer protein (PP). At the initiation of DNA replication, a hetero dimer complex with its own DNA polymerase and the PP supposed to interact with the terminal protein (TP), which is covalently bound to the template DNA (TP-DNA). PP contained an important adhesive amino acid sequence, Arg-Gly-Asp (RGD), near the carboxyl terminal. We have recently showed that the synthetic RGD peptide inhibited the transfection of phage M2. By site-directed mutagenesis, we introduced different amino acid into the RGD site of PP. These altered PP decreased obviously the priming activity in vitro.     

Conformational transformations in the protein lattice of phage P22 procapsids.

During the packaging of double-stranded DNA by bacterial viruses, the precursor procapsid loses its internal core of scaffolding protein and undergoes a substantial expansion to form the mature virion. Here we show that upon heating, purified P22 procapsids release their scaffolding protein subunits, and the coat protein lattice expands in the absence of any other cellular or viral components. Following these processes by differential scanning calorimetry revealed four different transitions that correlated with structural transitions in the coat protein shells. Exit of scaffolding protein from the procapsid occurred reversibly and just above physiological temperature. Expansion of the procapsid lattice, which was exothermic, occurred after the release of scaffolding protein. Partial denaturation of coat subunits within the intact shell structure was detected prior to the major endothermic event. This major endotherm occurred above 80 degrees C and represents particle breakage and irreversible coat protein denaturation. The results indicate that the coat subunits are designed to form a metastable precursor lattice, which appears to be separated from the mature lattice by a kinetic barrier.     

The tailspike protein of Shigella phage Sf6. A structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain

Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike of Salmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel beta-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel beta-helix protein with high structural similarity to its functional homolog from phage P22.