Small-angle X-ray scattering of reduced ribonuclease A: effects of solution conditions and comparisons with a computational model of unfolded proteins

The disulfide-reduced form of bovine ribonuclease A, with the Cys thiols irreversibly blocked, was characterized by small-angle x-ray scattering. To help resolve the conflicting results and interpretations from previous studies of this model unfolded protein, we measured scattering profiles using a range of solution conditions and compared them with the profiles predicted by a computational model for a random-coil polypeptide. Analysis of the simulated and experimental profiles reveals that scattering intensities at intermediate angles, corresponding to interatomic distances in the range of 5-20 Å, are particularly sensitive to changes in solvation and can be used to assess the internal scaling behavior of the polypeptide chain, expressed as a mass fractal dimension, D_m. This region of the scattering curve is also much less sensitive to experimental artifacts than is the very small angle regime (the Guinier region) that has been more typically used to characterize unfolded proteins. The experimental small-angle x-ray scattering profiles closely matched those predicted by the computational model assuming relatively small solvation energies. The scaling behavior of the polypeptide approaches that of a well-solvated polymer under conditions where it has a large net charge and at high urea concentrations. At lower urea concentrations and neutral pH, the behavior of the chain approaches that expected for θ-conditions, where the effects of slightly unfavorable interactions with solvent balance those of excluded volume, leading to scaling behavior comparable to that of an idealized random walk chain. Though detectable, the shift toward more compact conformations at lower urea concentrations does not correspond to a transition to a globule state and is associated with little or no reduction in conformational entropy. This type of collapse, therefore, is unlikely to greatly reduce the conformational search for the native state.     

(−)-Epigallocatechin-3-Gallate (EGCG) Maintains κ-Casein in Its Pre-Fibrillar State without Redirecting Its Aggregation Pathway

The polyphenol (−)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-β, α-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMκ-CN (reduced and carboxymethylated κ-casein) and thereby protect pheochromocytoma-12 cells from RCMκ-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMκ-CN [the IC₅₀ for 50 μM RCMκ-CN is 13 ± 1 μM]. Biophysical studies reveal that EGCG prevents RCMκ-CN fibril formation by stabilising RCMκ-CN in its native-like state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMκ-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent π-π stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated.     

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni²⁺) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.     

Antarctic bacterial haemoglobin and its role in the protection against nitrogen reactive species

In a cold and oxygen-rich environment such as Antarctica, mechanisms for the defence against reactive oxygen and nitrogen species are needed and represent important components in the evolutionary adaptations. In the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, the presence of multiple genes encoding 2/2 haemoglobins and a flavohaemoglobin strongly suggests that these proteins fulfil important physiological roles, perhaps associated to the peculiar features of the Antarctic habitat. In this work, the putative role of Ph-2/2HbO, encoded by the PSHAa0030 gene, was investigated by in vivo and in vitro experiments in order to highlight its involvement in NO detoxification mechanisms. The PSHAa0030 gene was cloned and then over-expressed in a flavohaemoglobin-deficient mutant of Escherichia coli, unable to metabolise NO, and the resulting strain was studied analysing its growth properties and oxygen uptake in the presence of NO. We here demonstrate that Ph-2/2HbO protects growth and cellular respiration of the heterologous host from the toxic effect of NO-donors. Unlike in Mycobacterium tuberculosis 2/2 HbN, the deletion of the N-terminal extension of Ph-2/2HbO does not seem to reduce the NO scavenging activity, showing that the N-terminal extension is not a requirement for efficient NO detoxification. Moreover, the ferric form of Ph-2/2HbO was shown to catalyse peroxynitrite isomerisation in vitro, confirming its potential role in the scavenging of reactive nitrogen species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom

This work reports the purification, biological characterization and amino acid sequence of two new basic PLA₂ isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA₂ were 14,175 ± 2.7 Da for Cdc-9 and 14,228 ± 3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca²⁺-binding and catalytic sites. The PLA₂ activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA₂ isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA₂ with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects.     

Reconstituted Micelle Formation Using Reduced, Carboxymethylated Bovine κ-Casein and Human β-Casein

In milk, κ-casein, a mixture of disulfide-bonded polymers, stabilizes and regulates the size of the unique colloidal complex of protein, Ca²⁺ and inorganic phosphate (P_i) termed the casein (CN) micelle. However, reduced, carboxymethylated bovine κ-CN (RCM-κ) forms fibrils at 37°C and its micelle-forming ability is in question. Here, the doubly- and quadruply-phosphorylated human β-CN forms and 1:1 (wt:wt) mixtures were combined with RCM-κ at different β/κ weight ratios. Turbidity (OD_{400 nm}) and a lack of precipitation up to 37°C were used as an index of micelle formation. Studies were with 0, 5 and 10 mM Ca²⁺ and 4 and 8 mM P_i. The RCM-κ does form concentration-dependent micelles. Also, β-CN phosphorylation level influences micelle formation. Complexes were low-temperature reversible and RCM-κ fibrils were seen. There appears to be equilibrium between fibrillar and soluble forms since the solution still stabilized after fibril removal. The RCM-κ stabilized better than native bovine κ-CN.     

Further characterization of the porcine LH subunits precursors in the translation products of pituitary RNAs

We have shown that the precursors of porcine LH α and β subunits have similar apparent molecular weights (Biochem. Biophys. Res. Comm., 1984, 118, 254). The aim of the present study was to characterize physiochemical features which permit to distinguish these precursors from each other. We report here that these precursors can be clearly distinguished by both their tryptic digests analyzed by one-dimension electrophoresis and their behavior in two-dimension electrophoresis whose first dimension was made in a non equilibrium pH gradient (2D-NEPHGE). The 2D-NEPHGE method developed in this study appears also useful to estimate simultaneously the translation activities of pituitary hormone mRNAs without a previous immunoprecipitation.     

Superior disintegrating properties of calcium cross-linked Cassia fistula gum derivatives for fast dissolving tablets

The present investigation was aimed at evaluating the feasibility of using calcium salts of carboxymethylated (CaCOG) or carbamoylethylated (CaCEG) derivatives of Cassia fistula gum as superdisintegrant while formulating fast disintegrating tablets (FDTs) exhibiting lowest disintegration time (DT) at highest mechanical strength. FDTs prepared with CaCOG (5%, w/w) or CaCEG (5%, w/w) were directly compressible and showed superior disintegrating property due to decreased water sorption time, increased particle packaging index, without any significant change in swelling index and effective pore radius. The mechanisms of superdisintegration suggested that probably the presence of Ca²⁺ resulted in intra and/or inter cross-linked bridges in the CaCOG or CaCEG that supported water transporting system even when the aqueous channels in the FDTs were blocked. Thus, the findings indicated great potential for using CaCOG or CaCEG as superdisintegrants in FDTs with high mechanical strength and low DT.     

Folding pathway of a circular form of bovine pancreatic trypsin inhibitor

The pathway of unfolding and refolding of a circular form of bovine pancreatic trypsin inhibitor, in which the termini were linked together in a peptide bond, has been examined by trapping and identifying the disulphide-containing intermediates, as was done previously for the unmodified protein. The folding pathway of the circular protein was essentially the same as that of the unmodified inhibitor, although there were differences in the distribution of intermediates that accumulated and in the rates of some steps. The effects of the cross-link between the termini on the stabilities of the folding intermediates and the native state were determined by measuring the rates of the interconversions making up the folding transition, and comparing them with those measured for the unmodified protein. The major effect of the cross-link was to stabilize an intermediate containing two native disulphides, (30-51, 14-38), but lacking the disulphide nearest the termini, 5-55. The native conformation was not measurably stabilized by the cross-link, in spite of the expected reduction of entropy of the unfolded state, indicating that the native state of the circular protein had a slightly strained conformation. The stabilities of the major one-disulphide intermediates were not significantly affected by the cross-link, suggesting that the termini of bovine pancreatic trypsin inhibitor do not tend to interact during the early stage of folding.