A simple model for proteins with interacting domains. Applications to scanning calorimetry data

Brandts, John F.; Hu, Cui Qing; Lin, Lung Nan; Mas, Maria T.

doi:10.1021/bi00447a048

Cited by 238 publications

(226 citation statements)

References 29 publications

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“…This suggests that domain interactions are weakened by reducing hydrophobic contacts or by placing a polar side chain into the hydrophobic interface. Further In the given context, Brandts et al (1989) developed a thermodynamic model that explains the stability of multi-domain proteins as the sum of increments of the separate domains plus contributions of the domain-domain interactions. Hence, if the less stable domain unfolds, its contribution to the stability of the more stable domain disappears.…”

Section: Discussionmentioning

confidence: 99%

“…According to the above model, which describes the stability of domain proteins as the sum of increments (Brandts et al, 1989), two extremes can be visualized: (i) Domains that unfold as independent entities will exhibit the same intrinsic stability, irrespective of whether they are isolated as separate "modules," or covalently linked (cf. Missiakas et al, 1990;Markovic-Housley et al, 1994); and (ii) if cooperative interactions between domains are essential for the intrinsic stability of the domains, folding and unfolding are strictly coupled so that the complete protein behaves as a single cooperative unit (cf.…”

Section: Discussionmentioning

confidence: 99%

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Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

1997

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AbstractyB-crystallin is a monomeric member of the By-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all+ Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one-or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner.It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete yB-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for yB wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of yB-crystallin.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

1997

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“…This result is contrary to what is generally observed for the unfolding of proteins with interacting domains. Theoretical models generally account for painvise domain-domain interactions by using a favorable interface free energy that goes to zero when one of the domains unfolds (Brandts et al, 1989;Freire et al, 1992). These models have been successful in explaining the unfolding properties of several proteins with domains communicating by painvise interactions.…”

Section: Discussionmentioning

confidence: 99%

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

et al. 1996

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Streptococcus equisirnilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al ., 1996, Protein Sci 5693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46°C and its stability is largely independent of the presence of the other domains. The hightemperatwe transition in intact SK (at approximately 63 "C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (AI and A2) adopt unstructured conformations when separated. AI binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

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“…As soon as one domain unfolds, the stabilizing effect of the complex formation vanishes, and, as a consequence, the second chain starts to unfold as well. For protein folding in cells, and thus for expression, however, the presence of one stably folded domain to serve as nucleation point for a less stable chain could be sufficient to substantially increase the amount of 36 In fact, studies have revealed that CH1 requires the interaction with the folded constant LC region controlling the assembly of the full-length antibody in vivo. 37 In addition, pairing of the variable domains increases the stability of the variable LC domain due to interface contacts.…”

Section: Discussionmentioning

confidence: 99%

Boosting antibody developability through rational sequence optimization

Seeliger

Schulz

Litzenburger

et al. 2015

mAbs

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(2015) Boosting antibody developability through rational sequence optimization , mAbs, 7:3, 505-515, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and longterm stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

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A simple model for proteins with interacting domains. Applications to scanning calorimetry data

Cited by 238 publications

References 29 publications

Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

Boosting antibody developability through rational sequence optimization

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