Probable localization of the bacteriophage T4 prehead proteinase zymogen in the center of the prehead core.

During the assembly of the bacteriophage T4 prehead, a T4-coded protease zymogen (P21) is built into the structure. At a certain stage in head formation, the protease precursor is activated and specifically cleaves most of the prehead proteins. In this paper we show that a correlation existed between the presence of proteinaceous material in the center of the prehead core, observed by electron microscopy, and the availability of P21 during prehead assembly. In the absence of P21, the core enclosed a hold of about 35 nm long and 20 nm wide. We found the same for (i) in vitro-assembled, negatively stained prehead-like structures and (ii) in vivo-formed preheads in thin sections of T4-infected cells. We concluded that P21 was localized in the center of the prehead core.     

Isolation and characterization of bacteriophage T4 mutant preheads.

To determine the function of individual gene products in the assembly and maturation of the T4 prehead, we have isolated and characterized aberrant preheads produced by mutations in three of the T4 head genes. Mutants in gene 21, which codes for the T4 maturation proteases, produce rather stable preheads whose morphology and protein composition are consistent with a wild-type prehead blocked in the maturation cleavages. Mutants in gene 24 produce similar structures which are unstable because they have gaps at all of their icosahedral vertices except the membrane attachment site. In addition, greatly elongated "giant preheads" are produced, suggesting that in the absence of P24 at the vertices, the distal cap of the prehead is unstable, allowing abnormal elongation of broth the prehead core and its shell. Vertex completion by P24 is required to allow the maturation cleavages to occur, and 24- preheads can be matured to capsids in vitro by the addition of P24. Preheads produced by a temperature-sensitive mutant in gene 23 are deficient in core proteins. We show that the shell of these preheads has the expanded lattice characteristic of the mature capsid as well as the binding sites for the proteins hoc and soc, even though none of the maturation cleavage takes place. We also show that 21- preheads composed of wild-type P23 can be expanded in vitro without cleavage.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

Functional empty capsid precursors produced by lambda mutant defective for late lambda DNA replication.

This report described lambda phage morphogenesis in a mutant system in which the normal pathways for late phage DNA (concatemer) synthesis are blocked and early (monomeric circular) DNA replication products accumulate. As shown earlier (Dawson et al., 1975) under these conditions, late proteins are synthesized and assembled into headlike structures. These structures that accumulate in the mutant are empty, suggesting the monomeric circular DNA molecules cannot be encapsulated. The present results show that crude extracts of induced lysogens of the mutant contain the complementation activities of preheads (the empty precursors to DNA-filled heads), tails, and DNA terminigenerating protein(s). Sucrose gradients of these crude extracts yield fractions containing prehead activity in relative amounts expected from the concentration of late proteins and empty structures. Furthermore, the proteins present in these fractions coelectrophorese with the known capsid proteins of preheads, and empty structures that look like preheads are observed in electron microscope examination of samples from the fractions. Based on our biological, biochemical, and electron microscope analyses, we conclude that the empty structures that accumulate in the induced lysogen of the mutant are normal preheads, which could become filled phage heads if DNA of the appropriate structure (i.e., "late DNA") were available.     

Assembly in vitro of the 50 S subunit from Escherichia coli ribosomes: proteins essential for the first heat-dependent conformational change.

An early intermediate during the assembly in vitro of the ribosomal 50 S subunit is the RI₅₀(1) particle, which undergoes a drastic change in s value to form the RI₅₀¹(1) particle. The formation of this RI₅₀¹(1) particle, which is essential for the assembly of a highly active 50 S particle, was analyzed. Total reconstitution experiments with purified proteins revealed that six ribosomal components (23 S RNA and the proteins L4, L13, L20, L22 and L24) are essential for the RI₅₀¹(1) formation. Protein L3 had a stimulatory effect, but was not essential. With these seven components, a particle with the RI₅₀¹ conformation could be formed.A comparison with assembly in vivo demonstrated that the pathways of protein assembly in vitro and in vivo are very similar at the beginning, but diverge towards the end of the process. Furthermore, the sequence in which the proteins can be split off the 50 S subunit by increasing concentrations of LiCl corresponds, to a first approximation, to the reverse of the order of assembly in vitro.     

SrmB,a DEAD-box helicase involved in Escherichia coli ribosome assembly,is specifically targeted to 23S rRNA in vivo

DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5′-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

Minimum Requirements for Bluetongue Virus Primary Replication In Vivo

The replication mechanism of bluetongue virus (BTV) has been studied by an in vivo reverse genetics (RG) system identifying the importance of certain BTV proteins for primary replication of the virus. However, a unique in vitro cell-free virus assembly system was subsequently developed, showing that it did not require the same set of viral components, which is indicative of differences in these two systems. Here, we studied the in vivo primary replicase complex more in-depth to determine the minimum components of the complex. We showed that while NS2 is an essential component of the primary replication stage during BTV infection, NS1 is not an essential component but may play a role in enhancing BTV protein synthesis. Furthermore, we demonstrated that VP7, a major structural protein of the inner core, is not required for primary replication but appears to stabilize the replicase complex. In contrast, VP3, the other major structural core protein, is an essential component of the complex, together with the three minor enzymatic proteins (VP1, VP4, and VP6) of the core. In addition, our data have demonstrated that the smallest minor protein, VP6, which is known to possess an RNA-dependent helicase activity, may also act as an RNA translocator during assembly of the primary replicase complex.     

In vitro polymerization of bacteriophage T4D tail core subunits

The in vitro polymerization of bacteriophage T4 core protein subunits (P19) onto baseplates is examined by electron microscopy. Procedures for purifying P19 and baseplates, both of which have in vitro complementation activity, are described. It is found that when P19 and baseplates are allowed to react over a wide range of initial concentration ratios, the most probable core length is about 1000 Å, the length found in vivo. Very few structures longer than this are observed if the polymerization is not allowed to proceed for much longer than one hour. Some physical properties of the baseplates were determined. These are s_20,W= 72 S, D_20,W = 8.56 × 10^?8 cm²/s, andM_r = 7.4 ± 0.3 × 10⁶.     

Synthesis of internal re-initiation fragments of beta-galactosidase in vitro and in vivo.

Internal re-initiation polypeptides which are products of the lacZ gene of Escherichia coli have been synthesized in a DNA-directed cell-free protein synthesis system. Some of the properties of these protein fragments have been characterized. The de novo synthesized re-initiation proteins, unlike both in vitro synthesized wild-type β-galactosidase and nonsense termination fragments, are insoluble when synthesized at 37 °C, but soluble if synthesis takes place at 25 °C. The same re-initiation proteins which are made in vivo have been detected in vitro. Unlike their in vivo counterparts, which are degraded rapidly, the in vitro synthesized restart proteins are completely stable for at least one hour following their synthesis. Both in vivo and in vitro, the re-initiation proteins are not synthesized from DNA containing a wild-type Z gene, but require a specific nonsense mutation in order to be expressed. Furthermore, the location of the mutation within the Z gene is very important in determining whether or not re-initiation will occur at a given site.Several nonsense mutations which do not result in the synthesis of detectable amounts of restart protein in vivo produce specific re-initiation polypeptides in vitro. These restart proteins display many of the same properties as do those which are made both in vivo and in vitro: they are not made from wild-type DNA, and they are only made from DNA containing a specific nonsense mutation. One of these mutations is 118, which is an extreme polar mutation in vivo. Another is 545, which synthesizes a restart protein in vivo if, and only if, it is coupled with a secondary mutation, π(1). π(1) thus appears to be necessary for the synthesis of a particular re-initiation protein in vivo but unnecessary for the synthesis of the same protein in vitro. The efficiencies of re-initiation vary at the different sites, but in all cases are less than the initiation frequency at the start of the gene. The experiments thus show that when complicating factors, such as polarity and protein degradation, are eliminated, translational re-initiation can be detected at many sites in the lacZ gene.     

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.     

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈ 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.     

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.     

Structure-based design and biochemical evaluation of sulfanilamide derivatives as hepatitis B virus capsid assembly inhibitors

Virus capsid structure is essential in virion maturation and durability, so disrupting capsid assembly could be an effective way to reduce virion count and cure viral diseases. However, currently there is no known antiviral which affects capsid inhibition, and only a small number of assembly inhibitors were experimentally successful. In this present study, we aimed to find hepatitis B virus (HBV) capsid assembly inhibitor which binds to the HBV core protein and changes protein conformation. Several candidate molecules were found to bind to certain structure in core protein with high specificity. Furthermore, these molecules significantly changed the protein conformation and reduced assembly affinity of core protein, leading to decrease of the number of assembled capsid or virion, both in vitro and in vivo. In addition, prediction also suggests that improvements in inhibition efficiency could be possible by changing functional groups and ring structures.     

Characterization of the In Vitro HIV-1 Capsid Assembly Pathway

During the morphogenesis of mature human immunodeficiency virus-1 cores, viral capsid proteins assemble conical or tubular shells around viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which capsid proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period, followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle preexisting tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature human immunodeficiency virus-1 core assembly and avenues for antiviral inhibition.     

Synthesis and degradation of termination and premature-termination fragments of beta-galactosidase in vitro and in vivo.

A number of abnormal polypeptides which are products of the lacZ gene in Escherichia coli have been characterized. A variety of experiments indicates that one class of at least nine different fragments arises from premature termination of protein synthesis. They have been detected as products of protein synthesis both in vitro and in vivo, although the percentage of fragments made in vitro is greater than that in vivo. The percentage of total Z gene-encoded protein which is found as fragments is estimated to be roughly 23% in vivo. This means that about 31% of the total number of β-galactosidase monomers synthesized are prematurely terminated molecules. The average molecular weight of the polypeptides produced is 91,000. This corresponds to a termination event occurring on the average once every 3200 codons. The sites at which termination occurs appear to be specific and are located primarily near the 3′-end of the gene.Polypeptides synthesized in vitro and in vivo from templates containing mutations in the Z gene have also been compared. Most of the mutationally generated fragments synthesized in vitro are stable, unlike their in vivo counterparts, which are often rapidly degraded. One fragment, however, generated by an early amber mutation in the Z gene, is degraded in the in vitro system. The mechanism of degradation appears to be specific for small abnormal polypeptides. Internal reinitiation polypeptides generated by nonsense mutations, which have been found in vivo, are not detected in the in vitro protein synthesis system under the conditions used here.     

Modulation of aggregation of silk fibroin by synergistic effect of the complex of curcumin and β-cyclodextrin

Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.     

Large self-assembled clathrin lattices spontaneously disassemble without sufficient adaptor proteins

Clathrin-coated structures must assemble on cell membranes to internalize receptors, with the clathrin protein only linked to the membrane via adaptor proteins. These structures can grow surprisingly large, containing over 20 clathrin, yet they often fail to form productive vesicles, instead aborting and disassembling. We show that clathrin structures of this size can both form and disassemble spontaneously when adaptor protein availability is low, despite high abundance of clathrin. Here, we combine recent in vitro kinetic measurements with microscopic reaction-diffusion simulations and theory to differentiate mechanisms of stable vs unstable clathrin assembly on membranes. While in vitro conditions drive assembly of robust, stable lattices, we show that concentrations, geometry, and dimensional reduction in physiologic-like conditions do not support nucleation if only the key adaptor AP-2 is included, due to its insufficient abundance. Nucleation requires a stoichiometry of adaptor to clathrin that exceeds 1:1, meaning additional adaptor types are necessary to form lattices successfully and efficiently. We show that the critical nucleus contains ~25 clathrin, remarkably similar to sizes of the transient and abortive structures observed in vivo. Lastly, we quantify the cost of bending the membrane under our curved clathrin lattices using a continuum membrane model. We find that the cost of bending the membrane could be largely offset by the energetic benefit of forming curved rather than flat structures, with numbers comparable to experiments. Our model predicts how adaptor density can tune clathrin-coated structures from the transient to the stable, showing that active energy consumption is therefore not required for lattice disassembly or remodeling during growth, which is a critical advance towards predicting productive vesicle formation.     

IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.