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Higher order iminodiacetic acid diamide trimer (560 compounds) and tetramer libraries (1260 compounds) are described and were assembled in a convergent multistep solution-phase synthesis for use in studying protein-protein interactions.
The reaction of [ReX(CO)5] with thiosemicarbazones H2L(R) derived from beta-keto esters (X = Cl, Br; R = Me, Ph) allowed the isolation of cyclic trimeric complexes [Re3(pyz(R))3(CO)9], where pyz(R) is the pyrazolonate corresponding to the thiosemicarbazone. Electron spray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) monitoring of the reactions of H2L(Ph) in toluene confirmed that the trimer was formed in the reaction mixture and no higher oligomer was detected. These studies, together with the X-ray structures of the trimeric complexes, afford new insight into the factors influencing the self-assembly of pyrazolonaterhenium(I) complexes.
Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.
The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, c (forming a disulfide-linked trimer), and d ("monomer"). Additional structural chains, "linkers," are required for the assembly of the approximately 200-polypeptide molecule. The proportion of linker chains had been reported to be one-third of the total mass on the basis of densitometry of Coomassie Blue-stained SDS-gels. Reverse-phase high performance liquid chromatography (HPLC), however, gave 16.3% linkers on the basis of both 220-nm absorbance and amino acid analysis (Ownby, D. W., Zhu, H., Schneider, K., Beavis, R. C., Chait, B. C., and Riggs, A. F. (1993) J. Biol. Chem. 268, 13539-13547). The subunit proportions have now been redetermined by SDS capillary electrophoresis as a test of the HPLC results. The electrophoresis, monitored at 214 nm, avoided the use of Coomassie Blue and provided results identical with those obtained by HPLC. Capillary electrophoresis monitored at both 214 and 415 nm was used to show that linker chains do not bind heme. Heme content has been found to be 2.9% by determination of hemin, amino acid analysis and dry weight. Measurement of the rate of hemin loss from oxidized L. terrestris Hb shows that high rates of loss can account for values of heme content significantly below 2.9% (or 0.26% iron).
Tripartite motif 5alpha (TRIM5alpha) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting the cells of particular species. TRIM5alpha is a member of the TRIM family of proteins, which contain RING, B-box, coiled-coil (CC), and, in some cases, B30.2(SPRY) domains. Here we investigated the abilities of domains from TRIM proteins (TRIM6, TRIM34, and TRIM21) that do not restrict HIV-1 infection to substitute for the domains of rhesus monkey TRIM5alpha (TRIM5alpha(rh)). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5alpha(rh) domains, allowing HIV-1 restriction. By contrast, similar chimeras containing the components of TRIM21, a slightly more distant relative of TRIM5, did not restrict HIV-1 infection. The TRIM21 B-box 2 domain and its flanking linker regions contributed to the functional defectiveness of these chimeras. All of the chimeric proteins formed trimers. All of the chimeras that restricted HIV-1 infection bound the assembled HIV-1 capsid complexes. These results indicate that heterologous RING, B-box 2, and CC domains from related TRIM proteins can functionally substitute for TRIM5alpha(rh) domains.
Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.
Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response. Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex. MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells. The cells presenting non-self peptides are cleared by CD8 positive T cells. In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated. Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.
The MHC class II-associated invariant chain (Ii) plays a central role in the biological function of MHC class II molecules. Ii is a type II membrane glycoprotein that is synthesized as different isoforms that include a major 31 kDa isoform (p31/p33) and a minor 41 kDa isoform (p41) in both, mice and humans. All isoforms share several common regions acting at different steps in the process that lead to functional class II molecule/peptide complexes. In the ER, two C-terminal extracytoplasmic regions of Ii are required for class II assembly: the 153-183 region is involved in the formation of Ii trimers and the 80-104 region mediates binding with class II molecules giving rise to nonamers. Ii association with class II molecules prevents both aggregation of class II dimers and binding with endogenous ER-derived peptides. In addition, two motifs in the cytosolic N-terminal region of Ii direct class II nonamers toward specialized endosomal compartments where peptide loading occurs. In these compartments, Ii undergoes proteolytic degradation leaving only CLIP (residues 80-104) associated with Class II. CLIP modulates loading of class II molecules in endosomes and is removed from the MHC class II groove by monomorphic MHC class II molecules, H2-M or HLA DM, in mouse and human, respectively. The roles of Ii in antigen presentation to MHC class II-restricted T cells and in CD4+ T cell development are discussed in this review.
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.
NEMO/IkappaB kinase (IKK) gamma is the regulatory component of the IKK complex comprising the two protein kinases, IKKalpha and IKKbeta. To investigate the self-assembly properties of NEMO and to understand further the mechanism of activation of the IKK complex, we purified wild-type and mutant NEMO expressed in Escherichia coli. In the absence of its IKK partners, recombinant NEMO (rNEMO) is a metastable functional monomer correctly folded, according to its fluorescence and far-UV CD spectra, which is binding specifically to the IKK complex. A minor fraction of rNEMO was found tightly associated with DnaK (E. coli Hsp70). We also examined the interaction of NEMO with prokaryotic and eukaryotic Hsp70, and we showed that the Hsp70-NEMO complex forms a supramolecular structure probably corresponding to an assembly intermediate. In vivo cross-linking experiments indicate that native NEMO in association with IKK is in equilibrium between a dimeric and a trimeric form. Similarly to native NEMO, a NEMO mutant deleted from its IKK binding N-terminal domain (residues 242-388) forms a stable trimeric coiled-coil, suggesting that the association of NEMO with IKK or with Hsp70 prevents incorrect interdomain pairing reactions that could lead to aggregation or to an non-native oligomeric state of rNEMO. We propose a model in which the activation of the IKK complex occurs through the trimerization of NEMO upon binding to a not yet identified upstream activator.
Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.
In nanotechnology, biomolecular assemblies serve not only as model systems for the construction of nanodevices, but they can also be used directly as templates for the formation of nanostructures. Biological nano-building blocks can either be isolated as complete functional units from living cells or viruses (biological "Top down" approach) or formed by biomolecular assembly from recombinant or synthetic components ("Bottom up" approach). In both cases, rational design of nanostructures requires knowledge of the stoichiometry of the biological structures, which frequently occur as multimers, i.e., the morphological complex is composed of multiple copies of one or more macromolecules. In this paper, a method is described for the stoichiometric quantification of molecules in bio-nanostructures. The method is based on using dilution factors and relative concentrations rather than absolute quantities, which are often difficult to determine, especially in short-lived assembly intermediates. The approach exploits the fact that the larger the stoichiometry of the component is, the more dramatic is the influence of the dilution factor (decrease in concentration) on the reaction. We established and used the method to determine the stoichiometry of components of bacterial virus phi29. The log of dilution factors was plotted against the log of reaction yield. The stoichiometry Z was determined with the equation Z=-1.58+2.4193T-0.001746T(2) [T in (0,1000), or 90 degree angle alpha in (0 degrees, 89.9 degrees )], where T is the slope of the curve (tangent of 90 degree angle alpha, which is the angle between the x-axis and the concentration dependent curve). Z can also be determined from a standard table given in this report. With the bacteriophage phi29 in vitro assembly system, up to 5x10(8) infectious virions per ml can be assembled from 11 purified components, giving our method a sensitivity of nine orders of magnitude. We confirmed the stoichiometries of phi29 components that were determined previously with microscopic approaches. The described method also responded to programmed stoichiometry changes, which were generated by assembling the phi29 DNA packaging motor from modified pRNA (DNA-packaging RNA) molecules forming a trimer of dimers or a dimer of trimers, instead of the wild-type hexamer.
Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.
Photosystem I (PSI) holocomplexes were fractionated to study the organization of the light-harvesting complex I (LHC I) pigment-proteins in barley (Hordeum vulgare) plastids. LHC Ia and LHC Ib can be isolated as oligomeric, presumably trimeric, pigment-protein complexes. The LHC Ia oligomeric complex contains both the 24- and the 21.5-kD apoproteins encoded by the Lhca3 and Lhca2 genes and is slightly larger than the oligomeric LHC Ib complex containing the Lhca1 and Lhca4 gene products of 21 and 20 kD. The synthesis and assembly of LHC I during light-driven development of intermittent light-grown plants occurs rapidly upon exposure to continuous illumination. Complete PSI complexes are detected by nondenaturing Deriphat (disodium N-dodecyl-[beta]-iminodipropionate-160)-PAGE after 2 h of illumination, and their appearance correlates with that of the 730- to 740-nm emission characteristic of assembled LHC I. However, the majority of the newly synthesized LHC I apoproteins are present as monomeric complexes in the thylakoids during the early hours of greening. We propose that during development of the protochloroplast the LHC I apoproteins are first assembled into monomeric pigmented complexes that then aggregate into trimers before becoming attached to the pre-existing core complex to form a complete PSI holocomplex.
A novel ordered assemblage of bacteriorhodopsin, a transmembrane protein functioning as a light-driven proton pump, is found in its three-dimensional crystal. Atomic force microscope images of the crystal surface reveal that spherical protein clusters with a diameter of approximately 50 nm are hexagonally close-packed. Electron micrographs of mechanically disintegrated crystals show that the inside of the protein cluster is filled with the mother liquor. The crystal is made up of hollow protein clusters. When disintegrated crystals are illuminated in the presence of a lipophilic anion, a significant alkalization of the external medium occurs. This result indicates that the protein cluster contains native lipids and that the cytoplasmic side of the protein faces the external medium. X-ray diffraction patterns and the observed diameter of the spherical shell suggest that approximately 200 bacteriorhodopsin trimers are aligned on a polyhedral surface lattice. Another remarkable feature of the spherical assemblies of bacteriorhodopsin is that they fuse with each other at low ionic strength and occasionally form a tubular or doughnut-like structure. The concept of membrane protein polymorphism is introduced on the basis of these observations, and it is used to describe the dynamic structure of some other biological membranes.
The folding of beta-structured, fibrous proteins is a largely unexplored area. A class of such proteins is used by viruses as adhesins, and recent studies revealed novel beta-structured motifs for them. We have been studying the folding and assembly of adenovirus fibers that consist of a globular C-terminal domain, a central fibrous shaft, and an N-terminal part that attaches to the viral capsid. The globular C-terminal, or "head" domain, has been postulated to be necessary for the trimerization of the fiber and might act as a registration signal that directs its correct folding and assembly. In this work, we replaced the head of the fiber by the trimerization domain of the bacteriophage T4 fibritin, termed "foldon." Two chimeric proteins, comprising the foldon domain connected at the C-terminal end of four fiber shaft repeats with or without the use of a natural linker sequence, fold into highly stable, SDS-resistant trimers. The structural signatures of the chimeric proteins as seen by CD and infrared spectroscopy are reported. The results suggest that the foldon domain can successfully replace the fiber head domain in ensuring correct trimerization of the shaft sequences. Biological implications and implications for engineering highly stable, beta-structured nanorods are discussed.
Bacteriorhodopsin (BR) and specific lipid molecules self-assemble into a quasi two-dimensional lattice structure known as the purple membrane (PM). In the PM, BR molecules exist in a trimeric form with lipid molecules present in the space enclosed by each trimeric unit and in the inter-trimer space. These trimeric units, which have a roughly circular cross-section, are arranged in hexagonal patterns with long-ranged crystalline order. In this work, we investigate the self-assembly of BR in the PM via Monte Carlo simulations of a two-dimensional model of the membrane and proteins. The protein molecules are modeled as 120 degrees sectors of a circle and the lipid molecules enter into the model through effective protein-protein interactions. The sectors cannot overlap with each other, and in addition to this excluded volume interaction there are site-site attractive interactions between specific points of the proteins to mimic interactions between helices on the proteins and lipid-induced interactions. At low values of the attractive well depth, the proteins are found in the monomeric form at all concentrations. At moderate and high values of the attractive well depth, trimers are formed as the concentration increases, and with a further increase in concentration the trimers organize into a hexagonal lattice. The interactions between the proteins and those induced by the intra-trimer lipids play an equally important role in the formation of trimers and the lattice. The lipids in the inter-trimer space cause the trimers to orient in a specific direction in the hexagonal crystal lattice.
Schmid metaphyseal chondrodysplasia results from mutations within the COOH-terminal globular domain (NC1) of type X collagen, a short chain collagen expressed in the hypertrophic region of the growth plate cartilage. Previous in vitro studies have proposed that mutations prevent the association of the NC1 domain of constituent chains of the trimer based upon a lack of formation of a trimeric structure that is resistant to dissociation with sodium dodecyl sulfate. To examine the effect of mutations on folding and assembly within a cellular context, bovine type X cDNAs containing analogous disease causing mutations Y598D, N617K, W651R, and wild-type were expressed in semi-permeabilized cells. We assessed trimerization of the mutant chains by their ability to form a collagen triple helix. Using this approach, we demonstrate that although there is an apparent lower efficiency of association of the mutant NC1 domains, they can drive the formation of correctly aligned triple helices with the same thermal stability as the wild-type collagen. When epitope-tagged mutant and wild-type collagen were co-expressed, heterotrimers could be detected by sequential immunoprecipitation. Both wild-type and mutant type X chains were found in association with the molecular chaperones protein disulfide isomerase and Hsp 47. The implications of these findings on the likely mechanism of Schmid metaphyseal chondrodysplasia will be discussed.