Biomedical subjects

P E Prevelige

Publications and source records attributed to P E Prevelige.

At least 19 recordsLinked to original sources

PubMed2001

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.

Bacteriophage P22↗

PubMed2001

Kinetic and calorimetric evidence for two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid.

A wide variety of viruses require the transient presence of scaffolding proteins to direct capsid assembly. In the case of bacteriophage P22, a model in which the scaffolding protein selectively stabilizes on-pathway growing intermediates has been proposed. The stoichiometry and thermodynamics of binding of the bacteriophage P22 scaffolding protein within the procapsid were analyzed by light scattering and isothermal titration calorimetry. Calorimetric experiments carried out between 10 and 37 degrees C were consistent with the presence of at least two distinct populations of binding sites, in agreement with kinetic evidence obtained by a light scattering assay. Binding to the high-affinity sites occurred at 20 degrees C with a stoichiometry of approximately 60 scaffolding molecules per procapsid and an apparent K(d) of approximately 100-300 nM and was almost completely enthalpy-driven. For the second binding population, precise fitting of the data was impossible due to small heats of binding, but the thermodynamics of binding were clearly distinct from the high-affinity phase. The heat capacity change (DeltaC(p)()) of binding was large for the high-affinity sites and negative for both sets of sites. Addition of sodium chloride (1 M) greatly reduced the magnitude of the apparent DeltaH, in agreement with previous evidence that electrostatic interactions play a major role in binding. A mutant scaffolding protein that forms covalent dimers (R74C/L177I) bound only to the high-affinity sites. These data comprise the first quantitative measurements of the energetics of the coat protein/scaffolding protein interaction.

Bacteriophage P22↗

PubMed2001

Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids.

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines.

Amino Acid Sequence↗

PubMed2001

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid.

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.

Amides↗

PubMed2000

Structural transformations accompanying the assembly of bacteriophage P22 portal protein rings in vitro.

The Salmonella typhimurium bacteriophage P22 assembles an icosahedral capsid precursor called a procapsid. The oligomeric portal protein ring, located at one vertex, comprises the conduit for DNA entry and exit. In conjunction with the DNA packaging enzymes, the portal ring is an integral component of a nanoscale machine that pumps DNA into the phage head. Although the portal vertex is assembled with high fidelity, the mechanism by which a single portal complex is incorporated during procapsid assembly remains unknown. The assembly of bacteriophage P22 portal rings has been characterized in vitro using a recombinant, His-tagged protein. Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time of approximately 1 h. Circular dichroic analysis of the monomers and rings indicated that the protein gained alpha-helicity upon polymerization. Thermal denaturation studies suggested that the rings contained an ordered domain that was not present in the unassembled monomer. A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is meta-stable and is susceptible to structural perturbation by bis-ANS. In conjunction with previously obtained data on the behavior of the P22 portal protein, we propose an assembly model for P22 portal rings that involves a meta-stable monomeric subunit.

Anilino Naphthalenesulfonates↗

PubMed2000

Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded DNA virus.

Scaffolding proteins are required for high fidelity assembly of most high T number dsDNA viruses such as the large bacteriophages, and the herpesvirus family. They function by transiently binding and positioning the coat protein subunits during capsid assembly. In both bacteriophage P22 and the herpesviruses the extreme scaffold C terminus is highly charged, is predicted to be an amphipathic alpha-helix, and is sufficient to bind the coat protein, suggesting a common mode of action. NMR studies show that the coat protein-binding domain of P22 scaffolding protein exhibits a helix-loop-helix motif stabilized by a hydrophobic core. One face of the motif is characterized by a high density of positive charges that could interact with the coat protein through electrostatic interactions. Results from previous studies with a truncation fragment and the observed salt sensitivity of the assembly process are explained by the NMR structure.

Amino Acid Sequence↗

PubMed2000

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy.

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.

Bacteriophage P22↗

PubMed2000

Identification of additional coat-scaffolding interactions in a bacteriophage P22 mutant defective in maturation.

Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation.

Bacteriophage P22↗

PubMed1999

Identification and characterization of the domain structure of bacteriophage P22 coat protein.

The bacteriophage P22 serves as a model for assembly of icosahedral dsDNA viruses. The P22 procapsid, which constitutes the precursor for DNA packaging, is built from 420 copies of a single coat protein with the aid of stoichiometric amounts of scaffolding protein. Upon DNA entry, the procapsid shell expands and matures into a stable virion. It was proposed that expansion is mediated by hinge bending and domain movement. We have used limited proteolysis to map the dynamic stability of the coat protein domain structures. The coat protein monomer is susceptible to proteolytic digestion, but limited proteolysis by small quantities of elastase or chymotrypsin yielded two metastable fragments (domains). The N-terminal domain (residues 1-180) is linked to the C-terminal domain (residues 205-429) by a protease-susceptible loop (residues 180-205). The two domains remain associated after the loop cleavage. Although only a small change of secondary structure results from the loop cleavage, both tertiary interdomain contacts and subunit thermostability are diminished. The intact loop is also required for assembly of the monomeric coat protein into procapsids. Upon assembly, coat protein becomes largely protease-resistant, baring cleavage within the loop region of about half of the subunits. Loop cleavage decreases the stability of the procapsids and facilitates heat-induced shell expansion. Upon expansion, the loop becomes protease-resistant. Our data suggest the loop region becomes more ordered during assembly and maturation and thereby plays an important role in both of these stages.

Bacteriophage P22↗

PubMed1999

Cavity defects in the procapsid of bacteriophage P22 and the mechanism of capsid maturation.

Bacteriophage P22 belongs to a family of double-stranded DNA viruses that share common morphogenetic features like DNA packaging into a procapsid precursor and maturation. Maturation involves cooperative expansion of the procapsid shell with concomitant lattice stabilization. The expansion is thought to be mediated by movement of two coat protein domains around a hinge. The metastable conformation of subunit within the procapsid lattice is considered to constitute a late folding intermediate. In order to understand the mechanism of expansion it is necessary to characterize the interactions stabilizing procapsid and mature capsid lattices, respectively. We employ pressure dissociation to compare subunit packing within the procapsid and expanded lattice. Procapsid shells contain larger cavities than the expanded shells, presumably due to polypeptide packing defects. These defects contribute to the metastable nature of the procapsid lattice and are cured during expansion. Improved packing contributes to the increased stability of the expanded shell. Comparison of two temperature-sensitive folding (tsf) mutants of coat protein (T294I and W48Q) with wild-type coat revealed that both mutations markedly destabilized the procapsid shell and yet had little effect on relative stability of the monomeric subunit. Thus, the regions affected by these packing defects constitute subunit interfaces of the procapsid shell. The larger activation volume of pressure dissociation observed for both T294I and W48Q indicates that the decreased stability of these particles is due to increase of cavity defects. These defects in the procapsid lattice are cured upon expansion suggesting that the intersubunit contacts affected by tsf mutations are absent or rearranged in the mature shell. The energetics of the in vitro expansion reaction also suggests that entropic stabilization contributes to the large free energy barrier for expansion.

Bacteriophage P22↗

PubMed1999

Solution x-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles.

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.

Bacteriophage P22↗

PubMed1999

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.

Bacteriophage P22↗

PubMed1998

Electrostatic interactions drive scaffolding/coat protein binding and procapsid maturation in bacteriophage P22.

The first step in assembly of the bacteriophage P22 is the formation of a T=7 icosahedral "procapsid," the major components of which are the coat protein and an inner core composed of the scaffolding protein. Although not present in the mature virion, the scaffolding protein is required for procapsid assembly. Eleven amino-acid residues at the extreme carboxyl terminus of the scaffolding protein are required for binding to the coat protein, and upon deletion of these residues, approximately 20 additional residues become disordered. Sequence analysis and NMR data suggest that the 30 residues at the carboxyl terminus form a helix-loop-helix motif which is stabilized by interhelical hydrophobic interactions. This "coat protein recognition domain" presents an unusually high number of positively charged residues on one face, suggesting that electrostatic interactions between this domain and the coat protein may contribute to recognition and binding. We report here that high ionic strength (1 M NaCl) completely inhibited procapsid assembly in vitro. When scaffolding protein was added to empty procapsid "shells" of coat protein, 1 M NaCl partially inhibited the binding of scaffolding protein to the shells. This suggests that the positively charged coat protein recognition domain at the carboxyl terminus of the scaffolding protein binds to a negatively charged region on the coat protein. During DNA packaging, the scaffolding protein exits the procapsid; scaffolding protein exit is followed by the expansion of the procapsid into a mature capsid. Procapsid shells can be induced to undergo a similar expansion reaction in vitro by heating (45-70 degreesC); this process was also inhibited by 1 M NaCl. These results are consistent with a model in which negatively charged scaffold protein-binding domains in the coat proteins move apart during procapsid expansion; this relief of electrostatic repulsion could provide a driving force for expansion and subsequent maturation. High-salt concentrations would screen this repulsion, while packaging of DNA (a polyanion) in vivo may increase the instability of the procapsid enough to trigger its expansion.

Bacteriophage P22↗

PubMed1998

Mechanism of capsid maturation in a double-stranded DNA virus.

Folding mechanisms of proteins incorporated within supramolecular assemblies, including viruses, are little understood and may differ fundamentally from folding mechanisms of small globular proteins. We describe a novel Raman dynamic probe of hydrogen-isotope exchange to investigate directly these protein folding/assembly pathways. The method is applied to subunit folding in assembly intermediates of the double-stranded DNA bacteriophage P22. The icosahedral procapsid-to-capsid maturation (shell expansion) of P22 is shown to be accompanied by a large increase in exchange protection of peptide beta-strands. The molecular mechanism of shell expansion involves unfolding of metastable tertiary structure to form more stable quaternary contacts and is governed by a surprisingly high activation energy. The results demonstrate that coat subunit folding and capsid expansion are strongly coupled processes. Subunit structure in the procapsid represents a late intermediate along the folding/assembly pathway to the mature capsid. Coupling of folding and assembly is proposed as a general pathway for the construction of supramolecular complexes.

Bacteriophage P22↗

PubMed1998

A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein.

The scaffolding protein of bacteriophage P22 directs the assembly of an icosahedral procapsid, a metastable shell that is the precursor for DNA packaging. The full-length protein has been shown previously to exist in a monomer-dimer-tetramer equilibrium of elongated and predominantly alpha-helical molecules. Two deletion-mutant fragments of the scaffolding protein, comprising amino acid residues 141 to 303 and 141 to 292, respectively, have been constructed, overexpressed in Escherichia coli, and purified. Removal of residues 1 to 140 yields a protein that is assembly-active both in vitro and in vivo, while the removal of the C-terminal 11 residues (293 to 303) leads to complete loss of scaffolding activity. Sedimentation analysis reveals that both scaffolding fragments exist in a monomer-dimer equilibrium governed by apparent dissociation constants Kd(141-303)=640 microM and Kd(141-292)=880 microM. Tetramer formation is not observed for either fragment; thus, the tetramerization domain of the scaffolding subunit resides in the N-terminal portion of the polypeptide chain. Examination of both fragments by circular dichroism, Raman and NMR spectroscopies indicates a highly alpha-helical fold in each case. Nonetheless, pronounced differences are observed between spectral signatures of the two fragments. Notably, Raman spectra of fragments 141-292 and 141-303 indicate that elimination of residues 293 to 303 results in unfolding of an alpha-helical coat protein "recognition" domain encompassing about 20 to 30 residues. The thermostability of fragment 141-303, monitored over a wide concentration range by circular dichroism and Raman spectroscopy, indicates a broad denaturation transition for the monomeric (low concentration) form, while more cooperative unfolding is observed for the dimeric (high concentration) form. A lesser increase in cooperativity upon dimerization is obtained for fragment 141-292. Additionally, the C-terminal recognition domain constitutes the most stable and cooperative unit in the 141-303 fragment. Measurement of hydrogen-isotope exchange kinetics in scaffolding fragments by time-resolved Raman spectroscopy shows that the C terminus is the only protected segment of the polypeptide chain. On the basis of the measured hydrodynamic and spectroscopic properties, a domain structure is proposed for the scaffolding subunit. The roles of these domains in P22 procapsid assembly are discussed.

Bacteriophage P22↗

PubMed1998

Functional domains of bacteriophage P22 scaffolding protein.

Assembly of the bacteriophage P22 requires a 303 amino acid residue scaffolding protein. Two scaffolding protein deletion mutants, consisting of residues 141 to 303 and 141 to 292, have been described. We report here that the 141-303 fragment, but not the 141-292 fragment, promoted procapsid assembly in vitro, bound to preformed shells of coat protein, and bound to a coat protein affinity column. These findings suggest that the carboxyl-terminal half of the scaffolding protein is sufficient for promoting assembly, and that the 11 amino acid residues at the extreme carboxyl terminus are required for binding to the coat protein. Analysis of the products of in vitro assembly reactions suggests that the maximum amount of scaffolding protein that can pack into a procapsid is dictated by the internal volume of the procapsid rather than by a finite number of binding sites. However, when the amount of scaffolding protein was reduced to limiting values, both the wild-type protein and the 141-303 fragment assembled procapsids with the same number, rather than the same mass, of scaffolding protein molecules. When the 141-292 fragment was added to a mixture of coat and scaffolding proteins, the initial phase of procapsid assembly was inhibited, but the final yield and composition of the procapsids were not affected. Assembly by a covalent dimeric mutant scaffolding protein (R74C/L177I) was not inhibited by the 141-292 fragment, which suggests that the inhibition is due to the formation of inactive heterodimers between the 141-292 fragment and the monomeric scaffolding protein. The 141-303 fragment, which has less tendency to self-associate than the wild-type protein, formed aberrant species as well as normal procapsid-like particles when the rate of assembly was high, suggesting that scaffolding protein dimerization may play a role in ensuring fidelity of assembly. Alternatively, residues 1 to 140 may play a direct structural role in preventing inappropriate scaffolding/coat protein interactions.

Bacteriophage P22↗

PubMed1998

Local rules simulation of the kinetics of virus capsid self-assembly.

A computer model is described for studying the kinetics of the self-assembly of icosahedral viral capsids. Solution of this problem is crucial to an understanding of the viral life cycle, which currently cannot be adequately addressed through laboratory techniques. The abstract simulation model employed to address this is based on the local rules theory of. Proc. Natl. Acad. Sci. USA. 91:7732-7736). It is shown that the principle of local rules, generalized with a model of kinetics and other extensions, can be used to simulate complicated problems in self-assembly. This approach allows for a computationally tractable molecular dynamics-like simulation of coat protein interactions while retaining many relevant features of capsid self-assembly. Three simple simulation experiments are presented to illustrate the use of this model. These show the dependence of growth and malformation rates on the energetics of binding interactions, the tolerance of errors in binding positions, and the concentration of subunits in the examples. These experiments demonstrate a tradeoff within the model between growth rate and fidelity of assembly for the three parameters. A detailed discussion of the computational model is also provided.

Biophysical Phenomena↗

PubMed1998

Inhibiting virus-capsid assembly by altering the polymerisation pathway.

Virus capsids assemble through the repeated interaction of well-defined protein subunits in a highly specific process. Basic research into the mechanism of protein polymerisation and virus assembly suggest that inhibition of the protein-protein interactions necessary for assembly is a valid therapeutic strategy. Computer models of virus-capsid assembly have located vulnerable stages in assembly, and small-molecule inhibitors of virus assembly have been identified. The challenge will be identifying agents that block assembly with the required specificity.

Capsid↗