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PubMed2003

Effect of colloidal carriers on ascorbyl palmitate stability.

Active compounds can be protected against degradation by incorporation into colloidal carrier systems. The stabilizing effect of carrier systems for ascorbyl palmitate (AP) was investigated using microemulsions (ME), liposomes and solid lipid nanoparticles (SLN). Analysis of chemical stability by HPLC showed that AP is most resistant against oxidation in non-hydrogenated soybean lecithin liposomes, followed by SLN, w/o and o/w ME, and hydrogenated soybean lecithin liposomes. The molecular environment of the AP-like nitroxide probe (C(16)-Tempo) in colloidal carriers was characterized using electron paramagnetic resonance (EPR) spectroscopy. We have found that the nitroxide groups are located in environments with different polarity and mobility. The hydrophilic part of AP is the reactive moiety, and high stability is obtained in systems in which this part is exposed to a less polar environment. Additionally, the determined accessibility of nitroxide groups to reduction correlated well with the chemical stability of AP. It is more deeply immersed in the interface when entrapped in a liquid-state carrier than when applied in gel-state particles. Encapsulation of AP in SLN core leads to greater stability. We conclude that the location of the sensitive group of the drug-molecule in a carrier system is crucial for its stability.

Ascorbic Acid↗
PubMed2001

Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase.

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Acylation↗
PubMedDate not supplied

Stabilization of (90)y-labeled DOTA-biomolecule conjugates using gentisic acid and ascorbic acid.

Radiolytic degradation of radiolabeled compounds is a major challenge for the development of new therapeutic radiopharmaceuticals. The goal of this study is to explore the factors influencing the solution stability of a (90)Y-labeled DOTA-peptide conjugate (RP697), including the amount of total activity, the activity concentration, the stabilizer concentration, and the storage temperature. In general, the rate of radiolytic decomposition of RP697 is much slower at the lower activity concentration (<4 mCi/mL) than that at the higher concentration (>10 mCi/mL). RP697 remains relatively stable at the 20 mCi level and room temperature while it decomposes rapidly at the 100 mCi level under the same storage conditions. Radical scavengers, such as gentisic acid (GA) and ascorbic acid (AA), were used in combination with the low temperature (-78 degrees C) to prevent the radiolytic decomposition of RP697. It was found that RP697 remains stable for at least 2 half-lives of (90)Y when GA or AA (10 mg for 20 mCi of (90)Y) is used as a stabilizer when the radiopharmaceutical composition is stored at -78 degrees C. The stabilizer (GA and AA) can be added into the formulation either before or after radiolabeling. The post-labeling approach is particularly useful when the use of a large amount of the stabilizer interferes with the radiolabeling. The radiopharmaceutical composition developed in this study can also apply to other (90)Y-labeled DOTA-biomolecule conjugates. The amount of the stabilizer used in the radiopharmaceutical composition and storage temperature should be adjusted according to the sensitivity of the radiolabeled DOTA-biomolecule conjugate toward radiolytic decomposition.

Ascorbic Acid↗
PubMed1993

Probing the determinants of disulfide stability in native pancreatic trypsin inhibitor.

The effects of single amino acid replacements on the stability of the 14-38 disulfide bond in the native form of bovine pancreatic trypsin inhibitor (BPTI) were measured. A total of 17 mutant proteins, with substitutions at one of 7 residues located 5-15 A from the disulfide in the native wild-type protein, were examined. The replacements were found to decrease the thermodynamic stability of the disulfide, as measured by exchange with thiol-disulfide reagents, by 0.6-5 kcal/mol, corresponding to a range of nearly 100 mV in redox potentials. The effects of the substitutions on disulfide stability were roughly correlated with the changes in side-chain volume, suggesting that optimal packing is a major factor in determining the stability of the disulfide in the wild-type protein. With only one exception, the substitutions also led to increases, as large as 50-fold, in the rates of disulfide reduction by dithiothreitol. The increased rates of reduction suggest that at least a fraction of the mutational destabilization of the disulfide is due to strain in the native protein that is relieved in the transition state for reduction. The stability of the disulfide in a peptide corresponding to the segments that are linked together by the 14-38 disulfide in native BPTI was found to be about 5 kcal/mol less than that of the disulfide in the intact wild-type protein. Together, the results with the mutant proteins and the peptide indicate that the stability of the disulfide in the native protein depends on both the local environment of the disulfide and on the ability of the rest of the protein to favor a conformation that promotes disulfide formation.

Amino Acid Sequence↗
PubMed1993

Effect of Na+ and nucleotide on the stability of solubilized Ca(2+)-free Ca-ATPase from scallop sarcoplasmic reticulum.

In membranous scallop sarcoplasmic reticulum, the alkali metal cations Na+ and K+ and nucleotide together promote dimer formation by the Ca(2+)-free Ca-ATPase and stabilize the enzyme activity [Kalabokis, V. N., Bozzola, J. J., Castellani, L., & Hardwicke, P. M. D. (1991) J. Biol. Chem. 266, 22044-22050]. The dependence of stabilization of the Ca(2+)-free membranous scallop Ca-ATPase on Na+ concentration does not show saturation and may involve several superimposed effects. In order to assess the contribution of dimer toward stabilization, i.e., determine the relative importance of intra- and intermolecular effects on stabilization, the influence of varying Na+ concentration and nucleotide on the decay of enzyme activity of the Ca(2+)-free detergent-solubilized Ca-ATPase was studied. Loss of enzyme activity on removal of Ca2+ with EGTA was associated with loss of capacity for phosphorylation by ATP, a Ca(2+)-dependent function. Stabilization of the soluble Ca(2+)-free enzyme by Na+ showed major differences from that seen with the membranous enzyme. The extent of stabilization of the Ca(2+)-free soluble enzyme by Na+ showed clear saturation with increasing Na+ concentration. In contrast to the Ca(2+)-free membranous enzyme, which is inactivated at pH 7.0 with biphasic first-order kinetics, loss of enzymatic function by the solubilized Ca-ATPase at pH 6.92, 0 degrees C, followed monophasic first-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Acid stabilization of insulin.

The effect of pH on the conformational stability of insulin was studied. Surprisingly, the Gibbs free energy of unfolding increased approximately 30% by acidification. pH titration of insulin's conformational stability is described by a transition involving a single proton with an apparent pK(a) of 7.0. The acid stabilization of insulin's conformation was attributed to the protonation of histidine at position 5 on the B-chain (HB5) as determined by 1H-NMR of the histidines, selective amino acid alteration, and enthalpies of ionization. Further acidification (at least to pH 2) does not decrease the free energy of unfolding. A conformational change in the tertiary structure, as indicated by the near-UV circular dichroism spectrum, accompanies this change in stability. We propose that this acid stabilization of insulin is physiologically important in maintaining insulin stability in the acid environment of the secretory/storage granules of the beta-cell of the pancreatic islets of Langerhans.

Chemical Phenomena↗
PubMed1992

Contribution of hydrogen bonding to the conformational stability of ribonuclease T1.

For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1). When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed. Twelve mutants of RNase T1 [Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)] have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A. On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond. This estimate is in good agreement with results from several related systems. Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect. Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides.

Amino Acid Sequence↗
PubMed1992

Relationship between local and global stabilities of proteins: site-directed mutants and chemically-modified derivatives of cytochrome c.

The methionine 80 sulfur-heme iron bond of rat cytochrome c, whose stability is decreased by mutating the phylogenetically invariant residue proline 30 to alanine and increased when tyrosine 67 is changed to phenylalanine, recovers its wild-type characteristics when both substitutions are performed on the same molecule. Titrations with urea, analyzed according to the heteropolymer theory [Alonso, D. O. V., & Dill, K. A. (1991) Biochemistry 30, 5974-5985], indicate that both single mutations increase the solvent exposure of hydrophobic groups in the unfolded state, while in the double mutant this conformational perturbation disappears. Similar increases in solvent exposure of hydrophobic groups are observed when the sulfur-iron bond of the wild-type protein is broken by alkylation of the methionine sulfur, by high pH, or by binding the heme iron with cyanide. The compensatory effects of the two single mutations do not extend to the overall stability of the protein. The added loss of conformational stability due to the single mutations amounts to 7.3 kcal/mol out of the 9 kcal/mol representing the overall free energy of stabilization of the native conformation of the wild-type protein. The folded conformation of the doubly mutated protein is only 2 kcal/mol less stable than that of the wild type. These results indicate that the double mutant protein is able to retain the essential folding pattern of cytochrome c and the thermodynamic stability of the methionine sulfur-heme iron bond, in spite of structural differences that weaken the overall stability of the molecule.

Amino Acid Sequence↗
PubMed1990

Glycine to alanine substitutions in helices of glyceraldehyde-3-phosphate dehydrogenase: effects on stability.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken was expressed in and purified from Escherichia coli. To investigate the physical basis of possible protein stabilization strategies, the effect of substitutions of glycine residues by alanine in helical regions was determined. One Gly to Ala substitution (G316A) located in the central core of the subunit was found to strongly stabilize the protein, while the other mutations are neutral or destabilize the protein. The effect seen for the stabilizing mutant in irreversible heat denaturation correlates with the first transition in folding equilibrium experiments that is observable by fluorescence, but not with the one detected by circular dichroism measurements or in dilution-induced dissociation experiments. The stabilizing effect of a Gly to Ala substitution therefore does not seem to be caused by an entropic effect on the unfolded state. Rather, an internal cavity is filled by the substitution G316A, probably stabilizing the native state. In large oligomeric proteins, imperfect packing may be a frequent cause of limited stability.

Alanine↗
PubMed1981

Protein-protein interactions: nature of the electrostatic stabilization of deoxyhemoglobin tetramer formation.

The summed electrostatic free energy contributions to deoxyhemoglobin A0 tetramer formation were computed at a series of pH and ionic strength values as the difference between the computed values for the tetramer and for the sum of the four individual chains. The electrostatic stabilization of each monomer is similar and close to that for myoglobin. At ionic strength 0.10 M the electrostatic contribution to the stability of the tetramer is approximately 35 kcal/mol at pH 6.0 and 18 kcal/mol at pH 9.6. The specific contribution to the stabilization of the tetramer, (sigma delta G"i,el)tet, is obtained by difference and shows a broad plateau above 7 kcal/mol over the range from pH 6.0 to 8.0, which is nearly obliterated by pH 9.6. By examination of the contributions of individual sites under the above summation, it is found that sites in the alpha chains are responsible for virtually the entire stabilizing effects in tetramer formation. The major differences on tetramer formation are sensed at eight sites. The stabilization provided by four of these sites results simply from changes in solvent exposure of sites in the given monomers as the tetramer is assembled. They are offset in part by changes at three sites that sense the greatest destabilization and that are responsible for the near cancellation of effects among the beta-chain sites. The general implications for the stabilization of molecular assemblies are considered.

Calorimetry↗
PubMed2001

Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: insight into the chaotrope unfolding dichotomy.

The balance between stabilizing forces and the localized electrostatic repulsions destabilizing the transthyretin (TTR) tetramer is tunable via anion shielding. The two symmetrical anion interaction sites in TTR are comprised of residues Lys15 and Lys15' from opposing subunits on the periphery of the two thyroxine binding sites. These epsilon-ammonium groups repel one another and destabilize the tetramer, unless an appropriate anion is present, which stabilizes the tetramer. Chaotrope denaturation of TTR exhibits unusual behavior in that urea appears to be a stronger denaturant than GdmCl (guanidinium chloride), even though GdmCl is typically twice as powerful as a denaturant. The shift in the midpoint of the urea denaturation curve to higher concentrations as well as the increase in the mole fraction of tetramer that is highly resistant to denaturation with increasing KCl concentration provides strong evidence that anion shielding stabilizes the TTR tetramer. A consequence of tetramer stabilization is folding hysteresis, because the high GdmCl concentrations required to denature the anion-stabilized tetramer do not allow refolding of the unfolded monomers. The formation of amyloid fibrils by TTR requires that its normal tetrameric structure dissociate to alternatively folded monomers, a process mediated by acidification (pH 5-4). This process is inhibited by Cl(-) ions in a concentration-dependent fashion. Chloride ion may not be the relevant physiological TTR stability modulator, but it is the main focus of these studies explaining the hysteresis observed in the denaturation and refolding studies with GdmCl.

Amino Acid Substitution↗
PubMed2002

Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer.

Glucose oxidase (GOD) from Aspergillus niger is a dimeric enzyme having high localization of negative charges on the enzyme surface and at the dimer interface. The monovalent cations induce compaction of the native conformation of GOD and enhance stability against thermal and urea denaturation [Ahmad et al. (2001) Biochemistry 40, 1947-1955]. In this paper we report the effect of the divalent cations Ca2+ and Mg2+ on the structural and stability properties of GOD. A divalent cation concentration dependent change in native conformation and subunit assembly of GOD was observed. Low concentration (up to 1 M) of CaCl2 or MgCl2 induced compaction of the native conformation of GOD, and the enzyme showed higher stability as compared to the native enzyme against urea denaturation. However, higher concentration (> or =2.0 M) of CaCl2 or MgCl2 induced dissociation of the native dimeric enzyme, resulting in stabilization of the enzyme monomer. An interesting observation was that the 3 M CaCl2-stabilized monomer of GOD retained about 70% secondary structure present in the native GOD dimer; however, there was a complete loss of cooperative interactions between these secondary structural elements present in the enzyme. Regarding the mechanism of divalent cation induced structural changes in GOD, the studies suggest that organization of water molecules by divalent cation results in stabilization of enzyme at low divalent cation concentration, whereas direct binding of these cations to the enzyme, at higher divalent cation concentration, results in dissociation and partial unfolding of the dimeric enzyme molecule.

Aspergillus niger↗
PubMed2003

Demonstration of two independently folding domains in the alpha subunit of bacterial luciferase by preferential ligand binding-induced stabilization.

The alpha subunit of bacterial luciferase unfolds and refolds reversibly by a three-state mechanism in urea-containing buffer. It has been proposed that the three-state unfolding of the alpha subunit arises from a stepwise unfolding of a C-terminal folding domain at lower concentrations of urea, followed by unfolding of the N-terminal domain at higher concentrations of urea (Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145). The location of an anion binding site in the proposed N-terminal folding domain allowed the folding mechanism to be probed in the context of the intact polypeptide. Anions preferentially stabilized the N-terminal domain in a concentration-dependent manner. The polyvalent anions sulfate and phosphate were found to be more stabilizing than monovalent chloride ion. Cations did not show a similar stabilizing effect, demonstrating that the stabilization was due to the anions alone. The purified N-terminal domain prepared by limited proteolysis and anion exchange chromatography was found to refold cooperatively with a midpoint approximately that of the second unfolding transition of the alpha subunit. Phosphate ion stabilized this fragment to roughly the same extent as it did the alpha subunit. The results presented are consistent with the proposed two-domain folding model and demonstrate that anion binding to the N-terminal folding domain stabilizes the alpha subunit of bacterial luciferase.

Anions↗
PubMed2005

Antisense recognition of the HER-2 mRNA: effects of phosphorothioate substitution and polyamines on DNA.RNA, RNA.RNA, and DNA.DNA duplex stability.

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.

Base Sequence↗
PubMed1996

Factors that affect the stabilization of alpha-helices in short peptides by a capping box.

It was reported recently that the capping box sequences of four N-terminal residues are very important for the stabilization of alpha-helices in proteins and peptides. To elucidate factors that affect the stabilization of alpha-helices in short peptides by this motif, we analyzed conformational properties of side chains of five N-terminal residues in several analogs of neuropeptide Y (NPY). The analysis revealed three previously unreported factors that appear to be important for stabilization of an alpha-helix: (a) a second capping box hydrogen bond for the side chains of Ser, Thr, and Cys; (b) long-range electrostatic interactions between the first (N-cap) and fifth (N4) residues; and (c) capping interactions of alpha-amino groups with the N4 residue. These factors were incorporated into the parameter set of a recently published, statistical mechanics approach that showed excellent accuracy in the prediction of the helical propensities of short peptides in water [Muñoz, V., & Serrano, L. (1995) J. Mol. Biol. 245, 275-296, 297-308]. A significant improvement in the agreement between theoretical predictions and experimental data was achieved. The present results also clarify the nature of capping box stabilization of alpha-helices in peptides and proteins, indicating that the total influence of hydrogen bonding, local interactions between side chains, helix macrodipole--charge/dipole interactions, and solvation possibilities must all be taken into account. All these factors are associated with approximately the same energy, but with different residues at the N-cap position, they may have opposite effects on the helix stability of peptides. Thus, a delicate balance of interactions of different types controls the stabilization properties of N-cap residues in alpha-helices.

Amino Acid Sequence↗
PubMed1996

Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein.

Efforts to enhance the stability of proteins by introducing engineered disulfide bonds have resulted in mixed success. Most approaches to the prediction of the energetic consequences of disulfide bond formation in proteins have considered only the destabilizing effects of cross-links on the unfolded state (chain entropy model) [Pace, C. N., Grimsley, G. R., Thomson, J. A., & Barnett, B. J. (1988) J. Biol. Chem. 263, 11820-11825: Doig, A. J., & Williams, D. H. (1991) J. Mol. Biol. 217, 389-398]. It seems clear, however, that disulfide bridges also can influence the stability of the native state. In order to assess the importance of the latter effect, we have studied four variants of staphylococcal nuclease (V8 strain) each containing one potential disulfide bridge created by changing two wild-type residues to cysteines by site-directed mutagenesis. In each case, one of the introduced cysteines was within the type VIa beta turn containing cis Pro117, and the other was located in the adjacent extended loop containing Gly79. In all four cases, the overall loop size was kept nearly constant (the number of residues in the loop between the two cysteines varied from 37 to 42) so as to minimize differences from chain entropy effects. The objective was to create variants in which a change in the reduction state of the disulfide would be coupled to a change in the position of the equilibrium between the cis and trans forms of the Xxx116-Pro117 peptide bond in the folded state of the protein. The position of this equilibrium, which can be detected by NMR spectroscopy, has been shown previously to correlate with the stability of the native protein. Its determination provides a measure of strain in the folded state. The thermal stabilities and free energies for unfolding by elevated temperature and guanidinium chloride were measured for each of the four mutants under conditions in which the introduced cysteines were cross-linked (oxidized) and unlinked (reduced). In addition, reduction potentials were determined for each mutant. Formation of the different disulfide bridges was found to induce varying levels of folded state strain. The stabilization energy of a given disulfide bridge could be predicted from the measured perturbation energy for the peptide bond isomerization, provided that energetic effects on the unfolded state were calculated according to the chain entropy model. Undiagnosed strain in native states of proteins may explain the variability observed in the stabilization provided by engineered disulfide bridges.

Disulfides↗
PubMed1997

Engineering the independent folding of the subtilisin BPN' prodomain: analysis of two-state folding versus protein stability.

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions. Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded. Here we manipulate the folding equilibrium of the unstable prodomain by introducing stabilizing mutations generated by design. By sequentially introducing three stabilizing mutations into the prodomain we are able to shift the equilibrium for independent folding from 97% unfolded to 65% folded. Spectroscopic and thermodynamic analysis of the folding reaction was carried out to assess the effect of stability on two-state behavior and the denatured state. The denatured states of single and combination mutants are not discernably different in spite of a range of DeltaGunfolding from -2.1 to 0.4 kcal/mol. Conclusions about the nature of the denatured state of the prodomain are based on CD spectral data and calorimetric data. Two state folding is observed for a combination mutant of marginal stability (DeltaG = 0). Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall DeltaGunfolding and the conformity of DeltaGunfolding vs T to two-state assumptions as embodied in the Gibbs-Helmholz equation. We believe our success in stabilizing the two-state folding reaction of the prodomain originates from the selection of mutations with improved ability to fold subtilisin rather than selection for increase in secondary structure content. The fact that a small number of mutations can stabilize the independent folding of the prodomain implies that most of the folding information already exists in the wild-type amino acid sequence in spite of the fact that the unfolded state predominates.

Bacillus subtilis↗
PubMed1998

A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.

Amino Acid Sequence↗