Hydration and protein folding in water and in reverse micelles: compressibility and volume changes. (49/513)

The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies.  (+info)

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins. (50/513)

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.  (+info)

Lifetimes of intermediates in the beta -sheet to alpha -helix transition of beta -lactoglobulin by using a diffusional IR mixer. (51/513)

The extremely slow alpha-helix/beta-sheet transition of proteins is a crucial step in amylogenic diseases and represents an internal rearrangement of local contacts in an already folded protein. These internal structural rearrangements within an already folded protein are a critical aspect of biological action and are a product of conformational flow along unknown metastable local minima of the energy landscape of the compact protein. We use a diffusional IR mixer with time-resolved Fourier transform IR spectroscopy capable of 400-micros time resolution to show that the trifluoroethanol driven beta-sheet to alpha-helix transition of beta-lactoglobulin proceeds via a compact beta-sheet intermediate with a lifetime of 7 ms, small compared with the overall folding time of beta-lactoglobulin.  (+info)

Conformational characterization of oligomeric intermediates and aggregates in beta-lactoglobulin heat aggregation. (52/513)

In one of the first studies of isolated intermediates in protein aggregation, we have used circular dichroism and fluorescence spectroscopy to characterize metastable oligomers that are formed in the early steps of beta-lactoglobulin heat aggregation. The intermediates show typical molten globule characteristics (secondary structure content similar to the native and less tight packing of the side chains), in agreement with the belief that partly folded states play a key role in protein aggregation. The far-UV CD signal bears strong resemblance to that of a known folding intermediate. Cryo-transmission electron microscopy of the aggregates reveals spherical particles with a diameter of about 50 nm and an internal threadlike structure. Isolated oligomers as well as larger aggregates bind the dye thioflavin T, usually a signature of the amyloid superstructures found in many protein aggregates. This result suggests that the structural motif recognized by thioflavin T can be formed in small oligomers.  (+info)

Isolation and characterization of casein mRNAs from lactating ewe mammary glands. (53/513)

RNA from bound polysomers of lactating ewe's mammary gland directs the synthesis of the three major milk proteins (alphas, beta and kappa-caseins) in a cell-free system derived from rabbit reticuyocytes. The "in vitro" product was identified by immunoprecipitation with specific antibodies and by electrophoresis in SDS polyacrylamide gel. Each of these messengers was purified from 20 to 25 fold from total membrane-bound polysomal RNA using poly U-Sepharose chromatography. This purified fraction assayed in a reticulocyte cell-free system is able to direct also the synthesis of 2 minor secretory proteins (beta-lactoglobulin and alpha-lactalbumin). The messenger RNAs purified by hybridization to poly U-Sepharose have a sedimentation coefficient of about 12 S and an apparent molecular weight of approximatively 3.5 s 10-5 daltons was observed by polyacrylamide gel electrophoresis under denaturing contitions. This value which correspond to about 900 nucleotides is significantly greater than the number expected for coding milk proteins.  (+info)

Dimerization, stability and electrostatic properties of porcine beta-lactoglobulin. (54/513)

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico-chemical characterization of porcine beta-lactoglobulin, purified from pooled milk: size-exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine beta-lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer-dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess alpha helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine beta-lactoglobulin in the crystal structure. Interaction studies revealed that porcine beta-lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.  (+info)

Interleukin-10-secreting Peyer's patch cells are responsible for active suppression in low-dose oral tolerance. (55/513)

We demonstrate the induction of antigen-specific interleukin-10 (IL-10)-secreting cells in murine Peyer's patches (PPs) after low-dose beta-lactoglobulin (BLG) feeding. In addition, we show that PP cells can inhibit the T-cell proliferative response in vitro as well as T-cell-mediated inflammation in vivo. The active suppression mediated by these regulatory cells was seen only within a narrow range of antigen dosage (feeding), with the most prominent effect at 5 x 1 mg BLG. On either side of this range, T-helper 1-like cytokine responses were observed when PP cells were stimulated with antigen in vitro. This result correlated with reduced production of regulatory cytokines as well as reduced activity of bystander suppression. We found that changes in IL-10 production correlated inversely with changes in interferon-gamma production. Inhibitory effects mediated by CD4(+) PP cells were partially neutralized by antibodies to IL-10 and transforming growth factor-beta. Interestingly, the generation of such regulatory cells after low-dose BLG feeding exhibited organ dependence. Among spleen, lymph node and PP cells derived from orally tolerized mice, PP cells were the most effective in promoting bystander suppression in the presence of BLG, indicating the significance of PPs as an inductive site for antigen-specific regulatory cells upon induction of low-dose oral tolerance. Moreover, PP cells from mice fed 5 x 1 mg BLG were shown to suppress a BLG-specific delayed-type hypersensitivity response induced in footpads, suggesting that IL-10-secreting PP cells regulate systemic inflammation.  (+info)

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane. (56/513)

Interactions between beta-lactoglobulin (beta-lg) and dimyristoylphosphatidylglycerol (DMPG) bilayers were studied using one- and two-dimensional infrared spectroscopy above (pD 7.4) and below (pD 4.4) the protein's (beta-lg's) isoelectric point (pI=5.2). The aim of the study was threefold: (1) gain a better understanding of beta-lg-phospholipid interaction; (2) provide information relative to the structure of beta-lg as it interacts with membranes; (3) determine whether the conformational modifications of the protein in the presence of lipids are strictly caused by thermal effects or whether they are modulated by the chain-melting phase transition. At pD 7.4, the lipid thermotropism, the acyl-chain order, and the membrane interfacial region were essentially unaffected by the presence of beta-lg, whereas the protein amide I region showed dramatic alterations. The results suggested the predominance of beta-sheets and alpha-helix elements, with a lost of structural integrity. At pD 4.4, beta-lg induced an approximately 2 degrees C downshift of the transition temperature, whereas the conformational order of the lipid chain decreased in the gel phase and increased in the liquid-crystalline phase. The hydration state of the DMPG C==O groups increased in the liquid-crystalline phase. The conformation of beta-lg at pD 4.4 in the presence of DMPG showed similarities with that observed at pD 7.4, but an increase in the alpha-helix content and a reduced thermal stability were noticed. In contrast to the protein alone, beta-lg aggregates in the presence of DMPG at pD 4.4 above 50 degrees C. At both pD values, the charged surface of the membrane seemed to be the main factor for inducing protein conformational changes by altering the intramolecular interactions that stabilize the native structure. However, protein incorporation within the membrane seemed to be involved at pD 4.4. The two-dimensional analysis performed with spectra recorded upon heating showed that spectral intensity changes at pD 4.4 and 7.4 occurred at the same frequencies in the amide I' region. The heat-induced structural changes of beta-lg were not correlated with the conformational modifications of the phospholipids along the phase transition, indicating that the thermal behavior of the protein was not modulated by the lipid chain melting, but rather represented the heat-induced protein rearrangement in the presence of DMPG.  (+info)