Irina I. Protasevich

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 (K a = 106 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.

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 Cys40.82 Ala 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.

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 approximated 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°C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71°C at pH 6.2 and 68°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.

Thermal Denaturation of Bacterial and Bovine Dihydrofolate Reductases and their Complexes with NADPH, Trimethoprim and Methotrexate

Scanning microcalorimetry was used for the study of thermal denaturation of E.coli and bovine liver dihydrofolate reductases (cDHFR and bDHFR, respectively) and their complexes with NADPH, trimethoprim (TMP) and methotrexate (MTX) at pH 6.8. It was shown that the denaturation temperature of bDHFR is 7.2 degrees C less than that of cDHFR and that ionic strength is equally important for the thermostability and cooperativity of the denaturation process of the two proteins. Binding of antifolate compounds significantly stabilizes DHFR against heat denaturation. The stabilizing effect and the transition cooperativity depend on the nature of the inhibitor, the presence of NADPH and the origin of the enzyme. The dependence of calorimetric denaturation enthalpy (calculated per gram of protein) on denaturation temperature for DHFRs, their complexes with NADPH and binary/ternary complexes with TMP/MTX fits to the same straight line with the slope of 0.66 J/Kg. This relatively high value indicates an essential role of hydrophobic contacts in the stabilization of DHFR structure. The change of denaturation temperatures in binary complexes with MTX/TMP (in comparison with the free enzymes) is as much as 14.2 degrees C/8.5 degrees C and 13.3 degrees C/3.2 degrees C for cDHFR and bDHFR, respectively. The same change in ternary complexes with MTX/TMP is much more pronounced and equals to 21.9 degrees C/16.8 degrees C and 29.0 degrees C/16.4 degrees C. The vast difference of binary and ternary complexes thermostability demonstrates the important role of cofactor in the stabilization of enzyme. Moving from binary to ternary systems causes a significant increase in denaturation temperatures, even when corresponding association constants do not change (cDHFR binary/ternary complexes with MTX) or increases very slightly (bDHFR binary/ternary complexes with TMP). In all other cases the increase of denaturation temperature for each protein in complex with ligands correlates with the association constant for the corresponding complex.

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 β-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°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.

Thermal stability of the polyheme cytochrome c3 superfamily

The cytochrome c 3 superfamily includes Desulfovibrio polyheme cytochromes c. We report the characteristic thermal stability parameters of the Desulfovibrio desulfuricans Norway (D.d.N.) cytochromes c 3 (M r 13,000 and M r 26,000) and the Desulfovibrio vulgaris Hildenborough (D.v.H.) cytochrome c 3 (M r 13,000) and high molecular mass cytochrome c (Hmc), as obtained with the help of electronic spectroscopy, voltammetric techniques and differential scanning calorimetry. The polyheme cytochromes are denatured over a wide range of temperatures: the D.v.H. cytochrome c 3 is highly thermostable (T d = 1°C) contrary to the D.d.N. protein (T d = 73 C). The thermostability of the polyheme cytochromes is redox state dependent. The results are discussed in the light of the structural and functional relationships within the cytochrome c 3 superfamily.

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.

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°C at pH 6.4 and 15.8°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°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.

Increased stability of human growth hormone with reduced lactogenic potency

Human growth hormone (hGH), whose main function is the somatic growth stimulation, induces diverse effects including lactation. We examined the possibility of hGH stabilization by elimination of its lactogenic activity. Chimeric GHs were constructed by replacement of different segments of hGH with sequences derived from non‐lactogenic porcine GH. As was observed in the rat Nb2‐11C lymphoma cell test, lactogenic activity of some chimeric hormones was seriously destroyed. This kind of hormones displayed the substantial increase in thermal and guanidine hydrochloride stability. The more stable hGH variants were found to be more soluble in Escherichia coli cells.

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.