Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1.
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Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes. (+info)
Amplification of the antibody response by C3b complexed to antigen through an ester link.
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Complement C3 has been described as playing an important role in the cell-mediated immune response. C3b has the capacity to covalently bind Ag and then to stimulate in vitro Ag presentation to T lymphocytes. To verify this observation in vivo, we prepared and purified covalent human C3b-Ag complexes using lysozyme (HEL) as Ag. The characterization of these HEL-C3b complexes indicates that they are representative of those susceptible to be generated in physiological conditions. Mice were immunized with 0.1 to 0.6 microgram of either free HEL, HEL + C3b, HEL-C3b, or HEL + CFA. Response was assessed after two i.p. injections by quantification of specific Ab production. Immunization with either HEL-C3b complexes or HEL + CFA leads to anti-HEL IgG production whereas free HEL or HEL + C3b was ineffective. Either HEL-C3b or HEL + CFA immunizations led to a similar Ig subclass patterns, including IgG1, IgG2a, IgA, and IgM. Our experiments provide the first evidence for modulation of specific Ab response by C3b when it is bound to Ag through a physiological-like link. Taken together with previous data concerning Ab response following recombinant HEL-C3d immunization, cellular events such as processing of C3b-Ag by APC and recognition by T lymphocytes, this present result underlines the importance of C3b and its fragments in stimulation of the immune system, through the multiplicity and complementarity of its interactions. (+info)
Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.
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The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment. (+info)
Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. I. Structure of the molecular complex.
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This paper reports on a simulation of a gramicidin channel inserted into a fluid phase DMPC bilayer with 100 lipid molecules. Two lipid molecules per leaflet were removed to insert the gramicidin, so the resulting preparation had 96 lipid molecules and 3209 water molecules. Constant surface tension boundary conditions were employed. Like previous simulations with a lower lipid/gramicidin ratio (Woolf, T. B., and B. Roux. 1996. Proteins: Struct., Funct., Genet. 24:92-114), it is found that tryptophan-water hydrogen bonds are more common than tryptophan-phospholipid hydrogen bonds. However, one of the tryptophan NH groups entered into an unusually long-lived hydrogen bonding pattern with two glycerol oxygens of one of the phospholipid molecules. Comparisons were made between the behavior of the lipids adjacent to the channel with those farther away. It was found that hydrocarbon chains of lipids adjacent to the channel had higher-order parameters than those farther away. The thickness of the lipid bilayer immediately adjacent to the channel was greater than it was farther away. In general, the lipids adjacent to the membrane had similar orientations to those seen by Woolf and Roux, while those farther away had similar orientations to those pertaining before the insertion of the gramicidin. A corollary to this observation is that the thickness of the hydrocarbon region adjacent to the gramicidin was much thicker than what other studies have identified as the "hydrophobic length" of the gramicidin channel. (+info)
Structural analysis of DNA-chlorophyll complexes by Fourier transform infrared difference spectroscopy.
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Porphyrins and metalloporphyrins are strong DNA binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. This study was designed to examine the interaction of calf thymus DNA with chlorophyll a (CHL) in aqueous solution at physiological pH with CHL/DNA(phosphate) ratios (r) of 1/160, 1/80, 1/40, 1/20, 1/10, and 1/5. Fourier transform infrared (FTIR) difference spectroscopy was used to characterize the nature of DNA-pigment interactions and to establish correlations between spectral changes and the CHL binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of DNA-CHL complexes in aqueous solution. Spectroscopic results showed that CHL is an external DNA binder with no affinity for DNA intercalation. At low pigment concentration (r = 1/160, 1/80, and 1/40), there are two major binding sites for CHL on DNA duplex: 1) Mg-PO2 and 2) Mg-N7 (guanine) with an overall binding constant of K = 1.13 x 10(4) M-1. The pigment distributions are 60% with the backbone PO2 group and 20% with the G-C base pairs. The chlorophyll interaction is associated with a major reduction of B-DNA structure in favor of A-DNA. At high chlorophyll content (r = 1/10), helix opening occurs, with major spectral alterations of the G-C and A-T bases. At high chlorophyll concentration (1/5), pigment aggregation is observed, which does not favor CHL-DNA complexation. (+info)
Crystal structure of human type II inosine monophosphate dehydrogenase: implications for ligand binding and drug design.
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Inosine monophosphate dehydrogenase (IMPDH) controls a key metabolic step in the regulation of cell growth and differentiation. This step is the NAD-dependent oxidation of inosine 5' monophosphate (IMP) to xanthosine 5' monophosphate, the rate-limiting step in the synthesis of the guanine nucleotides. Two isoforms of IMPDH have been identified, one of which (type II) is significantly up- regulated in neoplastic and differentiating cells. As such, it has been identified as a major target in antitumor and immunosuppressive drug design. We present here the 2.9-A structure of a ternary complex of the human type II isoform of IMPDH. The complex contains the substrate analogue 6-chloropurine riboside 5'-monophosphate (6-Cl-IMP) and the NAD analogue selenazole-4-carboxamide adenine dinucleotide, the selenium derivative of the active metabolite of the antitumor drug tiazofurin. The enzyme forms a homotetramer, with the dinucleotide binding at the monomer-monomer interface. The 6 chloro-substituted purine base is dehalogenated, forming a covalent adduct at C6 with Cys-331. The dinucleotide selenazole base is stacked against the 6-Cl-IMP purine ring in an orientation consistent with the B-side stereochemistry of hydride transfer seen with NAD. The adenosine end of the ligand interacts with residues not conserved between the type I and type II isoforms, suggesting strategies for the design of isoform-specific agents. (+info)
Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein.
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Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved eukaryotic protein that localizes predominantly in the mitochondrial matrix. It is thought to be involved in mitochondrial oxidative phosphorylation and in nucleus-mitochondrion interactions. We report the crystal structure of p32 determined at 2.25 A resolution. The structure reveals that p32 adopts a novel fold with seven consecutive antiparallel beta-strands flanked by one N-terminal and two C-terminal alpha-helices. Three monomers form a doughnut-shaped quaternary structure with an unusually asymmetric charge distribution on the surface. The implications of the structure on previously proposed functions of p32 are discussed and new specific functional properties are suggested. (+info)
Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization.
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We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer. (+info)