Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Sorbitol-mediated stabilization of human IgG against thermal inactivation

Sorbitol at 30% (w/w) stabilized human IgG to thermal denaturation from 60 to 85°C, increasing the protein's half life from 25 to 266 min at 70°C. A kinetic model based on the Lumry-Eyring inactivation scheme was developed and used to estimate the apparent rate constant and activation energy. © Rapid Science. 1998     

Using a second‐order differential model to fit data without baselines in protein isothermal chemical denaturation

In vitro protein stability studies are commonly conducted via thermal or chemical denaturation/renaturation of protein. Conventional data analyses on the protein unfolding/(re)folding require well‐defined pre‐ and post‐transition baselines to evaluate Gibbs free‐energy change associated with the protein unfolding/(re)folding. This evaluation becomes problematic when there is insufficient data for determining the pre‐ or post‐transition baselines. In this study, fitting on such partial data obtained in protein chemical denaturation is established by introducing second‐order differential (SOD) analysis to overcome the limitations that the conventional fitting method has. By reducing numbers of the baseline‐related fitting parameters, the SOD analysis can successfully fit incomplete chemical denaturation data sets with high agreement to the conventional evaluation on the equivalent completed data, where the conventional fitting fails in analyzing them. This SOD fitting for the abbreviated isothermal chemical denaturation further fulfills data analysis methods on the insufficient data sets conducted in the two prevalent protein stability studies.     

Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation

The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A _lim and k _I (A _lim is the limiting value of A at t and k _I is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant k _I calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A _lim and k _I may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.     

Protein unfolding and degradation by the AAA+ Lon protease

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.     

Multiple spectroscopic studies of the interaction between a quaternary ammonium-based cationic Gemini surfactant (as a carrier) and human erythropoietin

Erythropoietin (EPO) is a hematopoietic growth factor. This substance, as a strong cell protector, can increase cell maintenance during different damages of central nervous system. Since the brain-blood barrier prevents the entrance of large proteins similar to EPO into the brain, its systemic delivery gets limited. The aim of this study was to find an alternative approach for EPO delivery into the brain to skip the blood-brain barrier prevention. So, a new quaternary ammonium-based cationic Gemini surfactant has been used to study the interaction of the cationic Gemini surfactant (as a carrier) with EPO using various spectroscopic techniques of (fluorescence and circular dichroism (CD)) and thermal denaturation. Fluorescence spectroscopy studies show the formation of Gemini-EPO complex and also static quenching of protein upon this interaction. The binding parameters of number of binding sites, binding affinity, Gibbs free energy, enthalpy, and entropy were calculated according to fluorescence quenching studies. Also, CD results have further represented that the content of regular secondary structure of EPO did not show any significant alterations by increasing the Gemini concentration. Finally, thermal denaturation behavior of EPO results indicates decreasing the thermal stability of protein in the presence of Gemini. In conclusion, the obtained results proposed that Gemini as a cationic surfactant can bind to EPO without any significant diverse effects on the structure of this drug (EPO) which can be considered as a candidate for EPO delivery in future.     

Extracellular Poly(α-L-guluronate)lyase from Corynebacterium sp.: Purification, Characteristics, and Conformational Properties

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure—function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55°C, respectively. Metal compounds such as MnCl₂ and NiCl₂ increased the enzyme activity. The enzyme was identified as the endolytic poly(-L-guluronate)lyase, which was active on poly(-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the -form of the enzyme molecule and resembled poly(-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The -sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

Investigation of the folding pathway of the TEM-1 β-lactamase

The TEM-1 β-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 β-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley-Liss, Inc.