Thermodynamics of denaturation of complexes of barnase and binase with barstar

Mitkevich, Vladimir A.; Schulga, Alexey; Ermolyuk, Yaroslav S.; Lobachov, Vladimir M.; Chekhov, V. O.; Yakovlev, G. I.; Hartley, Robert W.; Pace, C. Nick; Kirpichnikov, Mikhaǐl P.; Makarov, Alexander А.

doi:10.1016/s0301-4622(03)00103-0

Cited by 22 publications

(19 citation statements)

References 14 publications

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“…Formation of the type II and III dimers partially or fully blocks the access to the active centre of binase for barstar and decreases the activity of binase versus the activity of barnase [17,19].…”

Section: Resultsmentioning

confidence: 99%

“…However, the activity of binase differs considerably from that of barnase [16,17]; the kinetic characteristics of the binase-barstar complex differ from the characteristics of barnase-barstar complex as well. It was demonstrated [18,19] that the association constant for the binase-barstar complex is by one order of magnitude lower than the constant for barnase inhibition by barstar.…”

Section: Introductionmentioning

confidence: 99%

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Brownian dynamics simulation of the competitive reactions: Binase dimerization and the association of binase and barstar

Ермакова

2007

Biophysical Chemistry

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Brownian dynamics simulation of the competitive reactions: Binase dimerization and the association of binase and barstar

Ермакова

2007

Biophysical Chemistry

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“…It is postulated that this association would be due to electrostatic interactions between positive charges in barnase/binase and negative ones in barstar. DSC studies show that mutations (leading to a loss in negative charge of the active site of barstar) in bartsarA decrease the affinity of the inhibitors for barnase and binase and increase the thermal stability of the barstar mutants [76]. Indeed these mutations reduce the electrostatic repulsion among the negatively charged groups at the binding site of barstar, leading to an increased stability and, reduce favourable interactions between the ribonucleases and the mutants.…”

Section: Dsc and Protein-ligand Interaction [67]mentioning

confidence: 99%

Differential Scanning Calorimetry in Life Science: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design

Bruylants¹,

Wouters²,

Michaux

2005

CMC

297

191

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All biological phenomena depend on molecular recognition, which is either intermolecular like in ligand binding to a macromolecule or intramolecular like in protein folding. As a result, understanding the relationship between the structure of proteins and the energetics of their stability and binding with others (bio)molecules is a very interesting point in biochemistry and biotechnology. It is essential to the engineering of stable proteins and to the structure-based design of pharmaceutical ligands. The parameter generally used to characterize the stability of a system (the folded and unfolded state of the protein for example) is the equilibrium constant (K) or the free energy (deltaG(o)), which is the sum of enthalpic (deltaH(o)) and entropic (deltaS(o)) terms. These parameters are temperature dependent through the heat capacity change (deltaCp). The thermodynamic parameters deltaH(o) and deltaCp can be derived from spectroscopic experiments, using the van't Hoff method, or measured directly using calorimetry. Along with isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) is a powerful method, less described than ITC, for measuring directly the thermodynamic parameters which characterize biomolecules. In this article, we summarize the principal thermodynamics parameters, describe the DSC approach and review some systems to which it has been applied. DSC is much used for the study of the stability and the folding of biomolecules, but it can also be applied in order to understand biomolecular interactions and can thus be an interesting technique in the process of drug design.

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“…Native wild‐type b* has cysteine residues at positions 40 and 82 that contribute to the stability of the bn–b* complex (Mitkevich et al 2003). They readily form adventitious inter‐ or intramolecular disulfide‐bonded species that do not inhibit barnase (Hartley 1993; Buckle et al 1994).…”

Section: Resultsmentioning

confidence: 99%

“…High resolution X‐ray structures of barnase–barstar complexes have only been reported for this cysteineless barstar and its mutants (Buckle et al 1994). The double alanine substitution results in an ∼10‐fold increase in K D (Hartley 1993; Mitkevich et al 2003). This Ψwt is also particularly advantageous for ESI–MS studies (see below).…”

Section: Resultsmentioning

confidence: 99%

Free energies of protein–protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods

2006

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The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solutionphase affinities of tightly bound protein-protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form proteinprotein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution-phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations <25 mM. Differences in DDG of dissociation between variants were determined with precisions <0.1 kcal/mol. The DDG values obtained deviate on average by 0.26 kcal/mol from those measured with a solution-phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein-protein-binding surfaces for complexes that have affinities in the picomolar to nanomolar range.

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Thermodynamics of denaturation of complexes of barnase and binase with barstar

Cited by 22 publications

References 14 publications

Brownian dynamics simulation of the competitive reactions: Binase dimerization and the association of binase and barstar

Brownian dynamics simulation of the competitive reactions: Binase dimerization and the association of binase and barstar

Differential Scanning Calorimetry in Life Science: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design

Free energies of protein–protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods

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