The Folding Pathway of 6aJL2.

One-third of the reported cases of light chain amyloidosis are related to the germ line λ6 family; remarkably, healthy individuals express this type of protein in just 2% of the peripheral blood and bone marrow B-cells. The appearance of the disease has been related to the inherent properties of this protein family. A recombinant representative model for λ6 proteins called 6aJL2 containing the amino acid sequence encoded by the 6a and JL2 germ line genes was previously designed and synthesized to study the properties of this family. Previous work on 6aJL2 suggested a simple two-state folding model at 25 °C; no intermediate could be identified either by kinetics or by fluorescence and circular dichroism equilibrium studies, although the presence of an intermediate that is populated at ∼2.4 M urea was suggested by size exclusion chromatography. In this study we employed classic equilibrium and kinetic experiments and analysis to elucidate the detailed folding mechanism of this protein. We identify species that are kinetically accessible and/or are populated at equilibrium. We describe the presence of intermediate and native-like species and propose a five-species folding mechanism at 25 °C at short incubation times, similar to and consistent with those observed in other proteins of this fold. The formation of intermediates in the mechanism of 6aJL2 is faster than that proposed for a Vκ light chain, which could be an important distinction in the amyloidogenic potential of both germ lines.

The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity

BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox-based modifications.

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

Insertion of antihypertensive peptides in acidic subunit from amaranth 11S induces contrasting effects in stability.

The insertion of peptides is a biotechnology tool widely used to improve the nutraceutical properties of proteins. Because the effect of these insertions in protein stability and function is difficult to predict, it should be determined experimentally. In this study, we created two variants of amarantin acidic subunit and analyzed them along with other four proteins reported previously. We measured their response against two destabilizing agents: temperature and urea. The six proteins presented the insertion of antihypertensive peptides (VYVYVYVY or RIPP) in the variable regions of the protein. We observed that their effect strongly depended on the site of the insertion. The insertion in the variable region I stabilized the protein both thermally and chemically, but it affected the inhibitory activity of the angiotensin-converting enzyme in vitro. In contrast, insertions in other three regions were severely destabilizing, producing molten globules. Our findings reveal that the insertion of bioactive peptides in variable regions of a protein can increase or decrease the protein's thermal and chemical stability and that these conformational changes may also alter its final activity.

Stability and aggregation propensity do not fully account for the association of various germline variable domain gene segments with light chain amyloidosis.

Variable domain (VL) gene segments exhibit variable tendencies to be associated with light chain amyloidosis (AL). While few of them are very frequent in AL and give rise to most of the amyloidogenic light chains compiled at the sequence databases, other are rarely found among the AL cases. To analyze to which extent these tendencies depend on folding stability and aggregation propensity of the germline VL protein, we characterized VL proteins encoded by four AL-associated germline gene segments and one not associated to AL. We found that the AL-associated germline rVL proteins differ widely in conformational stability and propensity to in vitro amyloid aggregation. While in vitro the amyloid formation kinetics of these proteins correlate well with their folding stabilities, the folding stability does not clearly correlate with their germline's frequencies in AL. We conclude that the association of the VL genes segments to amyloidosis is not determined solely by the folding stability and aggregation propensity of the germline VL protein. Other factors, such as the frequencies of destabilizing mutations and susceptibility to proteolysis, must play a role in determining the light chain amyloidogenicity.

Mutational and genetic determinants of λ6 light chain amyloidogenesis.

Approximately 25% of the λ6 light chains have glycine rather than arginine at position 25, which is an allelic variant of the IGLV6-57 (6a) locus. The Gly25 variant has been shown to decrease the folding stability of the germline λ6 V(L) protein 6aJL2 by 1.7 kcal·mol(-1). In this work, we compared the thermodynamic and fibrillogenic properties of the amyloidosis (AL) derived recombinant (r) V(L) protein AR, which contains the allelic variant Gly25, with those of germline rV(L) 6aJL2-R25G and the λ6 disease-associated V(L) proteins Wil (AL) and Jto (myeloma). Our experiments show that of the four proteins AR is the least stable; forms amyloid fibrils at physiological temperature, pH and ionic strength; has the shortest lag time; and elongates homologous seeds most efficiently. We conclude that the Gly25 allelic variant, together with the somatic mutations, contributes importantly to the extremely low stability and high amyloidogenicity of the AL-derived protein AR.

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains.

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

Thermodynamic and kinetic characterization of the association of triosephosphate isomerase: the role of diffusion.

It is known that diffusion plays a central role in the folding of small monomeric proteins and in the rigid-body association of proteins, however, the role of diffusion in the association of the folding intermediates of oligomeric proteins has been scarcely explored. In this work, catalytic activity and fluorescence measurements were used to study the effect of viscosity in the unfolding and refolding of the homodimeric enzyme triosephosphate isomerase from Saccharamyces cerevisiae. Two transitions were found by equilibrium and kinetic experiments, suggesting a three-state model with a monomeric intermediate. Glycerol barely affects DeltaG(0)(fold) whereas DeltaG(0)(assoc) becomes more favourable in the presence of the cosolvent. From 0 to 60% (v/v) glycerol, the association rate constant showed a near unitary dependence on solvent viscosity. However, at higher glycerol concentrations deviations from Kramers theory were observed. The dissociation rate constant showed a viscosity effect much higher than one. This may be related to secondary effects such as short-range glycerol-induced repulsion between monomers. Nevertheless, after comparison under isostability conditions, a slope near one was also observed for the dissociation rate. These results strongly suggest that the bimolecular association producing the native dimer is limited by diffusional events of the polypeptide chains through the solvent.

A comparative study of biochemical and immunological properties of triosephosphate isomerase from Taenia solium and Sus scrofa.

We produced the Taenia solium triosephosphate isomerase (TPI) in Escherichia coli and compared its biochemical and immunological properties with those of the commercial TPI from Sus scrofa. Taenia solium TPI is a homodimer composed of two 27-kDa monomers, with a specific activity of 5,683 U/mg and a Km value of 0.758, and S. scrofa TPI is also dimeric with similar monomeric molecular weight, specific activity of 4,227 U/mg, and a Km value of 0.51. The catalytic parameters for the isomerization of glyceraldehyde 3-phosphate, affinity between TPI monomers, and kinetic thermal denaturation and inactivation were similar for both enzymes. Anti-T. solium TPI antibodies cross-react weakly with Schistosoma mansoni TPI but do not cross-react with S. scrofa, human, or protozoan TPIs. These antibodies inhibited T. solium TPI activity but did not affect S. scrofa enzymatic activity. Immunizations with 1 microg of the T. solium TPI reduced 52% of cysticerci in a mouse-Taenia crassiceps model 1 mo after challenge. Our findings show that T. solium and S. scrofa TPIs possess similar biochemical and enzymatic properties but do not share immunological properties because anti-T. solium TPI antibodies did not recognize S. scrofa TPI. Inhibition of enzyme activity by anti-TPI antibodies suggests that they can be used as inhibitors of the enzyme.

Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica.

Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.