Vladimir M. Lobachov

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.

Natural polypeptides in left‐handed helical conformation A circular dichroism study of the linker histones’ C‐terminal fragments and β‐endorphin

Circular dichroism has been used to investigate the histone HI and H5 C‐terminal fragments and β‐endorphin conformation. It has been shown that in aqueous solution these polypeptides preferably adopt the left‐handed helical conformation of the poly‐l‐proline II type. A break in the linear temperature dependence of the CD value was found in the temperature interval between 50 and 55°C. It was proposed to be due to non‐cooperative disordering of the conformation caused by the destruction of the hydration shell.

Thermodynamics of denaturation of complexes of barnase and binase with barstar.

Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between -8 and -16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions.

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.

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.

Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves

Muscle of amphioxus contains large amounts of a four EF‐hand Ca2+‐binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N‐CaVP) and carboxy‐terminal halves (C‐CaVP). The interactive properties of recombinant and wild‐type CaVP are very similar, despite three post‐translational modifications in the wild‐type protein. N‐CaVP does not bind Ca2+, shows a well‐formed hydrophobic core, and melts at 44°C. C‐CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 μM (i.e., very similar to the entire CaVP). The metal‐free domain in CaVP and C‐CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+ forms melt in the 111°–123°C range, suggesting that C‐CaVP and the carboxy‐ domain of CaVP are natively unfolded in the metal‐free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy‐domain destabilize to different degrees the amino‐domain. Only C‐CaVP forms a Ca2+‐dependent 1:1 complex with CaVPT. Our results suggest that the carboxy‐terminal domain of CaVP interacts with CaVPT and that the amino‐terminal lobe modulates this interaction.

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.

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.