Structural features of single amino acid repeats in proteins.
(33/7757)
Single amino acid repeats are found in different kinds of proteins. Some of these repeats are pathogenic. It is striking that some amino acids are able to form such repeats, but other amino acids are not. We suggest an explanation for this fact based on the different tendency of each amino acid to form aggregates. Aggregation may be due to the formation of incipient lamellar crystals as they have been described in poly-alpha-amino acids and in most synthetic polymers. (+info)
Peptide recognition by two HLA-A2/Tax11-19-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures.
(34/7757)
The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11-19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11-19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides. (+info)
Purification of receptor complexes of interleukin-10 stoichiometry and the importance of deglycosylation in their crystallization.
(35/7757)
Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL-10 (vIL-10) is a homologue of human IL-10 (hIL-10) produced by Epstein-Barr virus (EBV). Both hIL-10 and vIL-10 bind to the soluble extracellular fragment of the cytokine receptor IL-10R1 (shIL-10R1). The stoichiometry of the vIL-10 : shIL-10R1 complex has been found to be the same as hIL-10 : shIL-10R1, with two vIL-10 dimers binding to four shIL-10R1 monomers. Complexes of both hIL-10 and vIL-10 with glycosylated shIL-10R1 could not be crystallized. Controlled deglycosylation using peptide : N-glycosidase F and endo-beta-N-acetylglucosaminidase F3 resulted in the formation of crystals of both hIL-10 : shIL-10R1 and vIL-10 : shIL-10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand-receptor complexes should facilitate our understanding of the basis of the interaction between IL-10 and the IL-10 receptor. (+info)
Nucleation of calcium oxalate crystals by albumin: involvement in the prevention of stone formation.
(36/7757)
BACKGROUND: Urine is supersaturated in calcium oxalate, which means that it will contain calcium oxalate crystals that form spontaneously. Their size must be controlled to prevent retention in ducts and the eventual development of a lithiasis. This is achieved, in part, by specific inhibitors of crystal growth. We investigated whether promoters of crystal nucleation could also participate in that control, because for the same amount of salt that will precipitate from a supersaturated solution, increasing the number of crystals will decrease their average size and facilitate their elimination. METHODS: Albumin was purified from commercial sources and from the urine of healthy subjects or idiopathic calcium stone formers. Its aggregation properties were characterized by biophysical and biochemical techniques. Albumin was then either attached to several supports or left free in solution and incubated in a metastable solution of calcium oxalate. Kinetics of calcium oxalate crystallization were determined by turbidimetry. The nature and efficiency of nucleation were measured by examining the type and number of neoformed crystals. RESULTS: Albumin, one of the most abundant proteins in urine, was a powerful nucleator of calcium oxalate crystals in vitro, with the polymers being more active than monomers. In addition, nucleation by albumin apparently led exclusively to the formation of calcium oxalate dihydrate crystals, whereas calcium oxalate monohydrate crystals were formed in the absence of albumin. An analysis of calcium oxalate crystals in urine showed that the dihydrate form was present in healthy subjects and stone formers, whereas the monohydrate, which is thermodynamically more stable and constitutes the core of most calcium oxalate stones, was present in stone formers only. Finally, urinary albumin purified from healthy subjects contained significantly more polymers and was a stronger promoter of calcium oxalate nucleation than albumin from idiopathic calcium stone formers. CONCLUSIONS: Promotion by albumin of calcium oxalate crystallization with specific formation of the dihydrate form might be protective, because with rapid nucleation of small crystals, the saturation levels fall; thus, larger crystal formation and aggregation with subsequent stone formation may be prevented. We believe that albumin may be an important factor of urine stability. (+info)
Temporal changes in mRNA expression for bikunin in the kidneys of rats during calcium oxalate nephrolithiasis.
(37/7757)
Inter-alpha-inhibitor and other bikunin-containing proteins are synthesized in relatively large quantities by the liver. These proteins function as Kunitz-type serine protease inhibitors and appear capable of inhibiting calcium oxalate (CaOx) crystallization in vitro. Preliminary studies have shown that renal tubular epithelial cells synthesize bikunin in response to CaOx challenge. To examine this response in vivo, a sensitive reverse transcription-quantitative competitive template-PCR was developed to detect and quantify poly(A)+ -tailed bikunin mRNA expression in kidney tissue from normal rats and rats developing CaOx nephrolithiasis after challenge with ethylene glycol. Bikunin mRNA expression in rat liver tissue was assessed as a positive control. The expression of bikunin mRNA in liver did not differ significantly between normal control rats and experimental rats with induced hyperoxaluria and renal CaOx crystallization. In contrast, there were significant temporal increases in the levels of bikunin mRNA expression in rat kidneys during CaOx nephrolithiasis after challenge with ethylene glycol. Urinary excretion of bikunin-containing proteins seemed to increase concomitantly. These findings indicate an association between the induction of hyperoxaluria/CaOx nephrolithiasis and the expression of the bikunin gene in rat kidneys. (+info)
Effect of glycerol on the interactions and solubility of bovine pancreatic trypsin inhibitor.
(38/7757)
The effects of additives used to stabilize protein structure during crystallization on protein solution phase behavior are poorly understood. Here we investigate the effect of glycerol and ionic strength on the solubility and strength of interactions of the bovine pancreatic trypsin inhibitor. These two variables are found to have opposite effects on the intermolecular forces; attractions increase with [NaCl], whereas repulsions increase with glycerol concentration. These changes are mirrored in bovine pancreatic trypsin inhibitor solubility where the typical salting out behavior for NaCl is observed with higher solubility found in buffers containing glycerol. The increased repulsions induced by glycerol can be explained by a number of possible mechanisms, all of which require small changes in the protein or the solvent in its immediate vicinity. Bovine pancreatic trypsin inhibitor follows the same general phase behavior as other globular macromolecules where a robust correlation between protein solution second virial coefficient and solubility has been developed. This study extends previous reports of this correlation to solution conditions involving nonelectrolyte additives. (+info)
Dehydration and crystallization of trehalose and sucrose glasses containing carbonmonoxy-myoglobin.
(39/7757)
We report a study wherein we contemporarily measured 1) the dehydration process of trehalose or sucrose glasses embedding carbonmonoxy-myoglobin (MbCO) and 2) the evolution of the A substates in saccharide-coated MbCO. Our results indicate that microcrystallization processes, sizeably different in the two saccharides, take place during dehydration; moreover, the microcrystalline structure is maintained unless the dry samples are equilibrated with a humidity >/=75% (>/=60%) at 25 degrees C for the trehalose (sucrose) sample. The evolution of the parameters that characterize the A substates of MbCO indicates that 1) the effects of water withdrawal are analogous in samples dried in the presence or in the absence of sugars, although much larger effects are observed in the samples without sugar; 2) the distribution of A substates is determined by the overall matrix structure and not only by the sample water content; and 3) the population of A0 substate (i. e., the substate currently put in relation with MbCO molecules having the distal histidine out of the heme pocket) is largely enhanced during the dehydration process. However, after rehumidification its population is largely decreased with respect to the values obtained, at similar water content, during the first dehydration run. (+info)
Structure and function of a membrane-bound murine MHC class I molecule.
(40/7757)
MHC molecules are expressed at the surface of nucleated cells to present peptides to T cells. Structural information on MHC molecules has been gathered by x-ray crystallography techniques by using soluble proteins. Although relationships between MHC molecules and cell membranes have not been studied in detail, they are of critical importance for T cell recognition. Using a chemically modified lipid, we have been able to capture and orient histidine-tagged MHC molecules on lipid membranes. Surface plasmon resonance experiments show that the protein binds to the nickel lipid in a specific manner and in an oriented fashion, which allows T cell receptor binding. Similar lipid surfaces have been used to grow two-dimensional crystals and to determine the structure of a membrane-anchored murine H-2Kb MHC class I molecule. The docking of the crystallographic structure into the three-dimensional reconstructed structure derived from the two-dimensional crystals allows us to determine that the histidine tag is near the membrane surface and that the MHC molecule is in an upright position, exposing the peptide/alpha1-alpha2 domains toward the T cell. (+info)