Differential scanning calorimetry of lobster haemocyanin

Guzmá‐Casado, Mercedes; Parody-Morreale, Antonio; Mateo, Pedro L.; Sánchez‐Ruiz, José M.

doi:10.1111/j.1432-1033.1990.tb15386.x

Cited by 56 publications

(27 citation statements)

References 30 publications

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“…In such cases, the thermal denaturation cannot be analysed using models that depend on the establishment of thermodynamic equilibria. Instead, kinetic models have to be used [39–43,53–62]. The present analysis confirms that the T m values of muscle and nonmuscle G‐actins depend on the heating rate, that the thermal unfolding proceeds with first‐order kinetics, and that it is an irreversible process (Scheme 1) [4,19,23,25,28].…”

Section: Discussionsupporting

confidence: 66%

Thermal unfolding of G‐actin monitored with the DNase I‐inhibition assay

Schüler

Lindberg

Schutt

et al. 2000

European Journal of Biochemistry

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Actin is one of the proteins that rely on chaperonins for proper folding. This paper shows that the thermal unfolding of G-actin, as studied by CD and ultraviolet difference spectrometry, coincides with a loss in DNase I-inhibiting activity of the protein. Thus, the DNase I inhibition assay should be useful for systematic studies of actin unfolding and refolding. Using this assay, we have investigated how the thermal stability of actin is affected by either Ca 2 1 or Mg 2 1 at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in different states of phosphorylation (ATP, ADP.P i , ADP.V i , ADP.AlF 4 , ADP.BeF x , and ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclude that the thermal unfolding of G-actin is a three-state process, in which an equilibrium exists between native actin with bound nucleotide and an intermediate free of nucleotide. Actins in the Mg-form were less stable than the Ca-forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle a-actin bovine cytoplasmic g-actin . yeast actin . cytoplasmic b-actin. The activation energies for the thermal unfolding reactions were in the range 200±290 kJ´mol 2 1 , depending on the bound ligands. Generally, the stability of the actin depended on the degree with which the nucleotide contributed to the connectivity between the two domains of the protein.

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Section: Discussionsupporting

confidence: 66%

Thermal unfolding of G‐actin monitored with the DNase I‐inhibition assay

Schüler

Lindberg

Schutt

et al. 2000

European Journal of Biochemistry

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“…These values not only compare well with each other and with that of a structurally and functionally related enzyme such as thermolysin, 282 f 8 kJ/mol [17], but are also close to that of a very structurally different protein such as hexameric haemocyanin, 383 30 kJ/mol [18].…”

Section: Procarboxypeptidase B and Carboxypeptidase Bsupporting

confidence: 60%

Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

Conejero‐Lara¹,

Sánchez‐Ruiz²,

Mateo³

et al. 1991

European Journal of Biochemistry

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High-sensitivity differential scanning calorimetry has been applied to the study of porcine pancreatic carboxypeptidase B, the proenzyme and its 81-residue activation domain. The thermal study has been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has been found to be reversible and corresponds to that of a typical compact globular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two-state irreversible model [Sanchez-Ruiz, J. M., L6pez-Lacomba, J. L., Cortijo, M. & Mateo, P. L. (1988) Biochemistry 27, 1648-16521. Thus the kinetic constants and activation parameters of the denaturation process could be obtained and compared to those for other proteins, particularly those of the closely related carboxypeptidase A system.The biological function of a protein depends on the correct folding of its native structure. The loss of this folded structure, either by heat or chemicals such as detergents, leads to an unfolded, inactive state. The subject of folding/unfolding and of protein thermal stability is currently a field of increasing interest because of biotechnological implications. For example, the efficiency of bioreactors can be greatly increased by using thermostable enzymes [l]. In addition, selected amino acid residue replacement in site-directed mutagenesis is often used to enhance protein stability [2 -41, while the structural characteristics responsible for thermostability in proteins from thermophilic organisms are also the object of current attention [5, 61.Differential scanning calorimetry (DSC) is nowadays the most useful technique for characterizing the thermal stability of proteins, quantitatively defined as the Gibbs energy of unfolding [7]. Thermodynamic analysis of DSC data for simple and complex proteins, which obviously requires the denaturation to be a reversible equilibrium process, has for instance led to the structural definition of co-operative submolecular domains [8]. Nevertheless, it is well known that the thermal denaturation of most proteins is calorimetrically irreversible. Since it is also generally accepted that the folded state is thermodynamically more stable than the unfolded one at low temperatures, additional irreversible processes must occur to the unfolded state to explain this overall irreversibility. Lumry and Eyring's scheme [9] is the simplest model to describe this situation N F ? U + F where N and U stand for the native and unfolded states, and F for the final state of the protein, which has been arrived at irreversibly. When the DSC transitions corresponding to irreversible protein denaturation are found to be highly scanrate-dependent, it is clear that the overall denaturation process is kinetically controlled an...

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“…The systematic analysis of in vitro , irreversible denaturation of proteins begun some 12 years ago and, after a significant number of experimental studies, a somewhat surprising picture has emerged. Thus, in many cases,18–46 irreversible thermal denaturation of proteins (as monitored by differential scanning calorimetry [DSC], e.g.) can be phenomenologically described on the basis of a simple two‐state irreversible model: in which only the native and final states are significantly populated and the conversion from N to F is determined by a strongly temperature‐dependent, first‐order rate constant ( k ap ).…”

Section: Introductionmentioning

confidence: 99%

Lower kinetic limit to protein thermal stability: A proposal regarding protein stability in vivo and its relation with misfolding diseases

2000

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In vitro thermal denaturation experiments suggest that, because of the possibility of irreversible alterations, thermodynamic stability (i.e., a positive value for the unfolding Gibbs energy) does not guarantee that a protein will remain in the native state during a given timescale. Furthermore, irreversible alterations are more likely to occur in vivo than in vitro because (a) some irreversible processes (e.g., aggregation, "undesirable" interactions with other macromolecular components, and proteolysis) are expected to be fast in the "crowded" cellular environment and (b) in many cases, the relevant timescale in vivo (probably related to the half-life for protein degradation) is expected to be longer than the timescale of the usual in vitro experiments (of the order of minutes). We propose, therefore, that many proteins (in particular, thermophilic proteins and "complex" proteins systems) are designed (by evolution) to have significant kinetic stability when confronted with the destabilizing effect of irreversible alterations. We show that, as long as these alterations occur mainly from non-native states (a Lumry-Eyring scenario), the required kinetic stability may be achieved through the design of a sufficiently high activation barrier for unfolding, which we define as the Gibbs energy barrier that separates the native state from the non-native ensemble (unfolded, partially folded, and misfolded states) in the following generalized Lumry-Eyring model: Native State <--> Non-Native Ensemble --> Irreversibly Denatured Protein. Finally, using familial amyloid polyneuropathy (FAP) as an illustrative example, we discuss the relation between stability and amyloid fibril formation in terms of the above viewpoint, which leads us to the two following tentative suggestions: (a) the hot spot defined by the FAP-associated amyloidogenic mutations of transthyretin reflects the structure of the transition state for unfolding and (b) substances that decrease the in vitro rate of transthyretin unfolding could also be inhibitors of amyloid fibril formation.

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Differential scanning calorimetry of lobster haemocyanin

Cited by 56 publications

References 30 publications

Thermal unfolding of G‐actin monitored with the DNase I‐inhibition assay

Thermal unfolding of G‐actin monitored with the DNase I‐inhibition assay

Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

Lower kinetic limit to protein thermal stability: A proposal regarding protein stability in vivo and its relation with misfolding diseases

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