Valine 108, a chain-folding initiation site-belonging residue, crucial for the ribonuclease A stability.

Thermal denaturation of bovine pancreatic ribonuclease A and a set of its single variants, carrying replacements of hydrophobic residues in the postulated 106-118 chain folding initiation site, has been studied by differential scanning calorimetry. Ribonuclease A variants undergo a two-state thermal transition denaturation except for those with replacement of valine 108. Most mutations cause a significant destabilization of the protein compared to the wild-type, thus demonstrating the importance of hydrophobic residues at the 106-118 region in maintaining the stability of the molecule. Among them, those of valine 108 promote the greatest (14-27 degrees C) destabilization of the molecule. Therefore, valine 108 plays a crucial role for ribonuclease A stability.

Apocalmodulin binds to the myosin light chain kinase calmodulin target site.

The interaction of a 20-residue-long peptide derived from the calmodulin-binding domain of the smooth muscle myosin light chain kinase with calcium-free calmodulin (apocalmodulin) was studied using a combination of isothermal titration calorimetry and differential scanning calorimetry. We showed that: (i) a significant binding between apocalmodulin and the target peptide (RS20) exists in the absence of salt (Ka = 10(6) M-1), (ii) the peptide interacts with the C-terminal lobe of calmodulin and adopts a partly helical conformation, and (iii) the presence of salt weakens the affinity of the peptide for apocalmodulin, emphasizing the importance of electrostatic interactions in the complex. Based on these results and taking into account the work of Bayley et al. (Bayley, P. M., Findlay, W.A., and Martin, S. R. (1996) Protein Sci. 5, 1215-1228), we suggest a physiological role for apocalmodulin.

Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar.

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.

Comparative study of monoclonal immunoglobulin M and rheumatoid immunoglobulin M by differential scanning microcalorimetry.

Thermal denaturation of monoclonal immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) and their Fab- and (Fc)5-fragments was studied by differential scanning microcalorimetry. The melting of IgM-RF started at a higher temperature than that of IgM and the maximum temperature of its main asymmetric peak of heat absorption was higher by 4 degrees C. At equal values of enthalpy, the thermal denaturation of IgM-RF and IgM consisted of four and five individual transitions, respectively, between pairs of states. The comparison of thermal denaturation parameters of Fab- and (Fc)5-fragments of IgM-RF and IgM showed a thermodynamic similarity of (Fc)5-fragments of both proteins, while their Fab-fragments differed in the interaction between VL-CL and VH-CH domains.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase.

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40,82Ala mutant are about 10(-12) M, only 5 to 43 times higher than those of the barnase-barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase-barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

Heat denaturation of pepsinogen in a water-ethanol mixture.

The effect of ethanol and pH on thermodynamic parameters and cooperativity of pepsinogen heat denaturation was studied by scanning microcalorimetry. Addition of 20% ethanol decreases the protein denaturation temperature by 10.7 degrees C at pH 6.4 and 15.8 degrees C at pH 8.0. It also decreases the denaturation heat capacity increment from 5.8 to 4.2 kcal/K.mol. The dependences of calorimetric denaturation enthalpy on denaturation temperature both in water and 20% ethanol are linear and intersect at about 95 degrees C. In 20% ethanol the pH shift from 5.9 to 8.0 results in a decreased number of cooperative domains in pepsinogen. This process causes no changes either in the secondary structure or in the local surroundings of aromatic amino acids. It is concluded that ethanol addition does not affect the cooperativity of pepsinogen denaturation substantially until the pH change provokes redistribution of charges in the protein molecule.

Thermostability of the barnase-barstar complex.

Scanning microcalorimetry was used to study heat denaturation of barnase in complex with its intracellular inhibitor barstar. The heat denaturation of the barnase-barstar complex is well approximately by two two-state transitions with the lower temperature transition corresponding to barstar denaturation and the higher temperature one to barnase denaturation. The temperature of barnase melting in its complex with barstar is 20 degrees C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71 degrees C at pH 6.2 and 68 degrees C at pH 8.0). It seems possible that when barstar unfolds it can remain bound to barnase, while the latter unfolds only on dissociation of the denatured barstar.

[The effect of ethanol on heat denaturation of pepsinogen]. / Vliianie étanola na teplovuiu denaturatsiiu pepsinogena.

The effect of ethanol and medium pH on thermodynamic parameters and cooperativity of pepsinogen thermal denaturation have been studied by scanning microcalorimetry. Addition of 20% (v/v) ethanol decreases the protein temperature of denaturation by 10.7 degrees C at pH 6.4 and by 15.8 degrees C at pH 8.0. It decreases the denaturation heat capacity change of pepsinogen from 5.8 to 4.2 kcal/K.mol, but has no effect on the number of energetic domains (regions melting in an "all-or-none" manner). The dependences of calorimetric denaturation enthalpy on denaturation temperature in both aqueous solution and 20% ethanol are linear and converge at about 95 degrees C, which coincides with the converge temperature of similar dependencies shown for a number of proteins in aqueous and water-alcohol solutions. A change of pH from 5.9 to 8.2 in 20% ethanol has been shown to cause a decrease of the number of cooperatively melting regions in pepsinogen. This process involves no changes either in the secondary structure or in the local surroundings of aromatic amino acids. It is concluded that ethanol addition has no effect on pepsinogen denaturation cooperativity until there is sufficient influence on intramolecular charge distribution, taking place upon a change of pH.

The studies of cooperative regions in T7 RNA polymerase.

The heat denaturation of bacteriophage T7 RNA polymerase (T7RNAP) was studied by scanning microcalorimetry. The thermodynamic parameters of the denaturation were estimated within the pH range 6-9. The analysis of the denaturation curves showed the presence of two cooperative parts of the T7RNAP molecule melting according to the 'all-or-none' principle. The molecular masses of these parts were determined as 22 and 77 kDa. These values are close to the molecular masses of protein domains obtained from X-ray diffraction and limited trypsinolysis data. The smaller N-terminal domain was shown to increase the thermostability of the 'catalytic' C-terminal domain within the intact T7RNAP molecule.

Comparative study of thermostability and structure of close homologues--barnase and binase.

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

[Comparison of the heat stability and structure close homologs--Bacillus amyloliquefaciens ribonuclease and Bacillus intermedius 7P ribonuclease]. / Sravneniee termostabil'nosti i struktury blizkikh gomologov-- ribonukleazy Bacillus amyloliquefaciens i ribonukleazy Bacillus intermedius 7P.

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase, in the pH region 2-6 have been determined. Barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as the pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp-94 by the His-18 residue, which is absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences have been found in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moment distribution.

Scanning microcalorimetry and circular dichroism study of melting of the natural polypeptides in the left-handed helical conformation.

It has been shown that in aqueous solution histone H1 and H5 C-terminal fragments and peptide hormones beta-endorphin and ACTH adopt preferably the left-handed helical conformation of the poly-L-proline II type. Scanning microcalorimetry and circular dichroism have been used to show that the linear temperature dependence of CD maximum amplitude and partial heat capacity value are broken in the temperature interval between 50 and 60 degrees C, after which [C]p reaches the constant level. It was proposed to be due to noncooperative disordering of the conformation caused by the destruction of the polypeptide hydration shell.

Effect of modification of carbohydrate component on properties of glucoamylase.

In this study, we investigated enzymatic deglycosylation of glucoamylase from Aspergillus awamori X 100/D27, a glycoprotein which has two N-linked and about forty short mannose-bearing O-linked sugars per molecule. O-Linked sugars were modified by treatment with alpha-mannosidase and N-linked sugars were removed using endo-beta-N-acetylglucosaminidase F. Analysis of conformational changes following deglycosylation suggests that O-linked sugars essentially contribute to the stabilization of glucoamylase domains. Modification of the carbohydrate component by adding 1-deoxymannojirimycin to the culture medium induced inhibition of alpha-mannosidases involved in the processing, leading to a more complete glycosylation and, consequently, to a higher stability of the enzyme.

[Extracellular alkaline ribonuclease of Bacillus thuringiensis var. subtoxicus]. / Vnekletochnaia shchelochnaia ribonukleaza Bacillus thuringiensis var. subtoxicus.

Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.

The number of cooperative regions (energetic domains) in a pepsin molecule depends on the pH of the medium.

The technique of scanning microcalorimetry was used to study the effect exerted by ethanol and by the pH of the medium on the number and size of cooperative regions in a pepsin molecule. Ethanol addition lowered the temperature of protein denaturation, but did not change the number of energetic domains. The number of thermodynamic cooperative units (determined as a delta Hcal to delta Heff ratio) was reduced from four to two when the pH changed from 6.7 to 2.0. As was demonstrated using the CD technique, this process involved no changes either in the secondary structure or in the local surroundings of aromatic amino acids. Therefore, variations in the cooperative properties of a pepsin globule at different pH values are associated with the electrostatic interactions of individual parts of the molecule.

[Number of cooperative regions (energy domains) in the pepsin molecule depends on the pH of the medium]. / Chislo kooperativnykh oblastei (énergeticheskikh domenov) v molekule pepsina zavisit ot pH sredy.

Ethanol and pH influence on the number and dimensions of cooperative regions in pepsin molecule was studied by scanning microcalorimetry. It is shown that ethanol solution causes a decrease of temperature of protein denaturation but does not influence the number of energetic domains. While changing pH from 6.7 to 2.0 the number of thermodynamic cooperative units (defined as the ratio delta Hcal/delta Heff) decreases from four to two. This process, as shown by CD technique, is followed by changes neither in the secondary structure, nor in the local environment of aromatic amino acids. A conclusion is made that the distinctions in cooperative characteristics of the protein globule at different pH are determined by electrostatic interactions of separate parts of the molecule.

[Microcalorimetric study of polypeptides in the conformation of the left helix of the poly-L-proline II type]. / Mikrokalorimetricheskoe issledovanie polipeptidov v konformatsii levoi spirali tipa poli-l-prolin II.

Microcalorimetry shows that polypeptide partial heat capacity changes linearly with temperature in the region of M-conformation existence and that there is a peculiarity in the transition region where this structure is exhausted, without cooperative heat absorption.