ABSTRACT
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 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (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 degrees -123 degrees 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.
Subject(s)
Calcium-Binding Proteins/chemistry , Muscle Proteins/chemistry , Amino Acid Sequence , Animals , Circular Dichroism , EF Hand Motifs/physiology , Escherichia coli/genetics , Protein Conformation , Protein Denaturation/physiology , Protein Folding , Protein Structure, Tertiary/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , ThermodynamicsABSTRACT
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.
Subject(s)
Ribonuclease, Pancreatic/chemistry , Animals , Calorimetry, Differential Scanning , Cattle , Circular Dichroism , Enzyme Stability , Escherichia coli/genetics , In Vitro Techniques , Models, Molecular , Mutagenesis, Site-Directed , Pancreas/enzymology , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribonuclease, Pancreatic/genetics , Ribonuclease, Pancreatic/metabolism , Thermodynamics , Valine/chemistryABSTRACT
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.
Subject(s)
Calmodulin/metabolism , Myosin-Light-Chain Kinase/metabolism , Calcium/metabolism , Calorimetry, Differential Scanning , Circular Dichroism , Protein Binding , Protein Conformation , ThermodynamicsABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Cysteine/metabolism , Endoribonucleases/metabolism , Ribonucleases/metabolism , Alanine/genetics , Bacillus/enzymology , Bacterial Proteins/genetics , Cysteine/genetics , Enzyme Stability , Heating , Mutagenesis, Site-Directed , Protein DenaturationABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Hot Temperature , Ribonucleases/metabolism , Bacterial Proteins/chemistry , Calorimetry, Differential Scanning , Enzyme Stability , Escherichia coli/enzymology , Hydrogen-Ion Concentration , Protein Denaturation , Ribonucleases/chemistry , ThermodynamicsABSTRACT
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.
Subject(s)
Adrenocorticotropic Hormone/chemistry , Protein Conformation , Protein Denaturation , beta-Endorphin/chemistry , Animals , Calorimetry, Differential Scanning , Cattle , Chickens , Circular Dichroism , Hot Temperature , Peptides/chemistry , Pituitary Gland, Anterior/chemistry , SwineABSTRACT
Circular dichroism has been used to investigate the histone H1 and H5 C-terminal fragments and beta-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 degrees C. It was proposed to be due to non-cooperative disordering of the conformation caused by the destruction of the hydration shell.
