Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations.
(57/15188)
Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly alpha-helical to beta-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native alpha conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in beta structure. The soluble beta form (beta-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to beta-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation. (+info)
Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin.
(58/15188)
Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic. (+info)
Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum.
(59/15188)
Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes. (+info)
The protein disulphide-isomerase family: unravelling a string of folds.
(60/15188)
The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities. Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins. Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members. (+info)
FORESST: fold recognition from secondary structure predictions of proteins.
(61/15188)
MOTIVATION: A method for recognizing the three-dimensional fold from the protein amino acid sequence based on a combination of hidden Markov models (HMMs) and secondary structure prediction was recently developed for proteins in the Mainly-Alpha structural class. Here, this methodology is extended to Mainly-Beta and Alpha-Beta class proteins. Compared to other fold recognition methods based on HMMs, this approach is novel in that only secondary structure information is used. Each HMM is trained from known secondary structure sequences of proteins having a similar fold. Secondary structure prediction is performed for the amino acid sequence of a query protein. The predicted fold of a query protein is the fold described by the model fitting the predicted sequence the best. RESULTS: After model cross-validation, the success rate on 44 test proteins covering the three structural classes was found to be 59%. On seven fold predictions performed prior to the publication of experimental structure, the success rate was 71%. In conclusion, this approach manages to capture important information about the fold of a protein embedded in the length and arrangement of the predicted helices, strands and coils along the polypeptide chain. When a more extensive library of HMMs representing the universe of known structural families is available (work in progress), the program will allow rapid screening of genomic databases and sequence annotation when fold similarity is not detectable from the amino acid sequence. AVAILABILITY: FORESST web server at http://absalpha.dcrt.nih.gov:8008/ for the library of HMMs of structural families used in this paper. FORESST web server at http://www.tigr.org/ for a more extensive library of HMMs (work in progress). CONTACT: [email protected]; [email protected]; [email protected] (+info)
The epidermal growth factor precursor. A calcium-binding, beta-hydroxyasparagine containing modular protein present on the surface of platelets.
(62/15188)
Various human body fluids and secretions contain a soluble form of the epidermal growth factor (EGF) precursor. The EGF precursor molecule contains eight EGF modules in addition to EGF itself. Using monoclonal antibodies specific for the EGF modules 7 and 8, we have purified the soluble form of the EGF precursor from human urine to homogeneity. The protein was shown to have a molecular mass of about 160 kDa and the N-terminal sequence SAPNHWSXPE. EGF modules 2, 7 and 8 of the precursor have the consensus sequence for post-translational beta-hydroxylation of Asp/Asn residues. We identified the presence of erythro-beta-hydroxy-aspartic acid (Hya) in acid hydrolysates of the EGF precursor (2.4 M.M protein-1). As the DNA sequence encodes Asn in the corresponding position, the Hya represents erythro-beta-hydroxyasparagine (Hyn). The Hyn-containing modules have a consensus calcium-binding motif immediately N-terminal of the first Cys residue. The synthetic EGF module 2 (residues 356-395) of the EGF precursor was found to bind calcium with low affinity, Kd approximately 3.5 mM, i.e. similar to the affinity of other isolated calcium-binding EGF modules. EGF module 7, when part of the intact protein, was found to bind Ca2+ with a Kd approximately 0.2 microM, i.e. approximately 10(4)-fold higher than that of isolated EGF modules presumably due to the influence of neighboring modules. We have detected EGF precursor in platelet-rich plasma and demonstrated it to be associated to platelets. The platelets were found to have 30-160 EGF molecules each. (+info)
Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A.
(63/15188)
The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments. (+info)
Importance of the carboxyl terminus in the folding and function of alpha-hemolysin of Staphylococcus aureus.
(64/15188)
The physical state of two model mutants of alpha-hemolysin (alphaHL), alphaHL(1-289), a carboxyl-terminal deletion mutant (CDM), and alphaHL(1-331), a carboxyl-terminal extension mutant (CEM), were examined in detail to identify the role of the carboxyl terminus in the folding and function of native alphaHL. Denatured alphaHL can be refolded efficiently with nearly total recovery of its activity upon restoration of nondenaturing conditions. Various biophysical and biochemical studies on the three proteins have revealed the importance of an intact carboxyl terminus in the folding of alphaHL. The CDM exhibits a marked increase in susceptibility to proteases as compared with alphaHL. alphaHL and CEM exhibit similar fluorescence emission maxima, and that of the CDM is red-shifted by 9 nm, which indicates a greater solvent exposure of the tryptophan residues of the CDM. In addition, the CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases its fluorescence intensity significantly unlike alphaHL and CEM, which show marginal binding. The circular dichroism studies point that the CDM possesses significant secondary structure, but its tertiary structure is greatly diminished as compared with alphaHL. These data show that the CDM has several of the features that characterize a molten globule state. Experiments with freshly translated mutants, using coupled in vitro transcription and translation, have further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the water-soluble form of alphaHL. The studies demonstrate a critical role of the carboxyl terminus of alphaHL in attaining the native folded state. (+info)