Enhancing Recombinant Protein Quality and Yield by Protein Stability Profiling

Mezzasalma, Tara M.; Kranz, James K.; Chan, Winnie; Struble, Geoffrey T.; Schalk-Hihi, Céline; Deckman, Ingrid C.; Springer, Barry A.; Todd, Matthew J.

doi:10.1177/1087057106297984

Cited by 79 publications

(58 citation statements)

References 32 publications

(54 reference statements)

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“…4 characterization of protein stability in the presence of various excipients 27 and the optimization of conditions for protein crystallization 28 .…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Zubrienė¹,

Gutkowska²,

Matulienė³

et al. 2010

Biophysical Chemistry

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Radicicol is a natural antibiotic that specifically inhibits chaperone Hsp90 activity and binds to its active site with nanomolar affinity. Radicicol has been widely used as a lead compound to generate synthetic analogs with reduced toxicity and increased stability that could be employed clinically. Here we present a detailed thermodynamic description of radicicol binding to human Hsp90 and yeast Hsc82 studied by isothermal titration calorimetry and thermal shift assay.Titrations as a function of pH showed a linked protonation event upon radicicol binding. The intrinsic binding constant and the thermodynamic parameters (including the enthalpy, entropy, and heat capacity) were determined for yeast Hsc82, and human alpha and beta Hsp90. Recent experimental evidence in literature shows that yeast Hsc82 has significant differences from human Hsp90 isozymes. Here we support this by demonstrating differences in radicicol binding thermodynamics to these proteins. The intrinsic enthalpy of radicicol binding to Hsc82 was -46.7 kJ/mol, to Hsp90alpha -70.7 kJ/mol, and to Hsp90beta was -66.8 kJ/mol. The enthalpies of binding were significantly different, while the intrinsic dissociation constants were quite similar,

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“…4 characterization of protein stability in the presence of various excipients 27 and the optimization of conditions for protein crystallization 28 .…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Zubrienė¹,

Gutkowska²,

Matulienė³

et al. 2010

Biophysical Chemistry

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“…Melting temperatures (T m ) were determined by monitoring the fluorescence change of ANS as it binds to the hydrophobic interior of proteins upon denaturation (Mezzasalma et al, 2007). GRK2 or GRK variants (0.05-0.2 mg/ml) were incubated at saturating ligand concentrations and 100 M ANS in a total volume of 10 l in triplicate, using ABgene 384-well polymerase chain reaction microtiter plates (Thermo Fisher Scientific, Waltham, MA).…”

Section: Downloaded Frommentioning

confidence: 99%

Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

Thal

Yeow

Schoenau³

et al. 2011

Mol Pharmacol

105

162

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G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G␤␥ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G␤␥ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

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“…The studies showed that Na/K phosphate buffer stabilized FMS by 1.5 degrees compared with HEPES buffer and imidazole and nickel destabilize the protein by 1.22 and 1.1 degrees at 100 mM and 0.1 mM, respectively. More details of FMS stability profiling are described elsewhere (56).…”

Section: Figure 1 Schematic View Of Fms Full-length Cytoplasmic Domamentioning

confidence: 99%

Protein Engineering of the Colony-stimulating Factor-1 Receptor Kinase Domain for Structural Studies

Schalk-Hihi

Struble

et al. 2007

Journal of Biological Chemistry

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A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.

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Enhancing Recombinant Protein Quality and Yield by Protein Stability Profiling

Cited by 79 publications

References 32 publications

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

Protein Engineering of the Colony-stimulating Factor-1 Receptor Kinase Domain for Structural Studies

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