Stabilization of Proteins by Ligand Binding:  Application to Drug Screening and Determination of Unfolding Energetics

Waldron, Travis T.; Murphy, Kenneth P.

doi:10.1021/bi034212v

Cited by 165 publications

(165 citation statements)

References 9 publications

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“…For proteins with multiple ligand-binding sites, P is expanded (SI Text). By thermodynamic linkage (3,30) any change in ΔG U caused predominantly by ligand binding (Eq. 1) is…”

Section: Resultsmentioning

confidence: 99%

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Isom

Vardy

Oas

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCRderived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics. Macromolecular stability is therefore one of the most fundamental thermodynamic measures in biochemistry by quantitatively reporting on structure-function relationships to provide a universal monitor for biochemical function.There are two distinct approaches for determining protein stability (4). The first measures the free energy of protein (un)folding under equilibrium conditions by assessing the fraction of the native state using spectroscopy, hydrodynamic observations, functional assays, or calorimetry. The second exploits the relationship between protein dynamics and stability by monitoring the differential reactivity of internal chemical groups in native and unfolded states. This second approach measures conformational free energies, which under appropriate conditions corresponds to global protein stability. Amide proton exchange is used most commonly to monitor such differential reactivity (5-9), but its widespread use to assess biological function typically is limited by the need for specialized instrumentation and relatively large amounts of protein. Recently, cysteine reactivity (10-14) and proteolysis (15) have emerged as alternative means to determine rates of protein (un)folding and estimate protein stabilities. Here we present a method, quantitative cysteine reactivity (QCR), in which protein stability is determined by monitoring the reactivity of cysteine residues buried in the hydrophobic core of proteins. This approach has the advantage over more traditional methods for measuring protein stability in that it requires only picomoles (nanograms) of protein, uses simple instrumentation accessible to any lab, can be reasonably high throughput, and can provide site-specific thermodynamic information. QCR can be used to determine apparent protein stabilities rapidly and accurately, construct Gibbs-Helmholtz stability profiles, measure ligand binding over a large range of ligand concentrations and affinities, and infer e...

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“…For proteins with multiple ligand-binding sites, P is expanded (SI Text). By thermodynamic linkage (3,30) any change in ΔG U caused predominantly by ligand binding (Eq. 1) is…”

Section: Resultsmentioning

confidence: 99%

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Isom

Vardy

Oas

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…S1). Such effects have previously been linked to entropic contributions in free energies of ligand association (23,32).…”

Section: Discussionmentioning

confidence: 99%

Gaining ligand selectivity in thyroid hormone receptors via entropy

Martı́nez

Nascimento

Nunes

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Triac ͉ design ͉ mobility N uclear receptors (NRs) are conditional transcription factors with important roles in development and disease (1, 2). Although these proteins are targets for existing drugs and pharmaceutical development, structural relationships between subsets of these proteins mean that ligands that target one NR can cross-react with others. This is commonly observed when NRs exist in multiple subtypes, as for thyroid hormone (TH)

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“…The increase in thermal stability (ΔT m ) can be related directly to the binding affinity (K d ) of ligands for the protein (75), but only when the association rate and binding enthalpy of the ligands are constant (64). These same principles of thermal stabilization can also be applied to protein·protein complexes (62,63).…”

Section: Discussionmentioning

confidence: 99%

“…Also, a phylogenetic tree of these RI proteins from diverse organisms ( Figure 5A) has an architecture and rate of evolution similar to that of the phylogenetic tree of ribonucleases from the same organisms ( Figure 5B) (52-54). To gain insight into the co-evolution of affinity of this femtomolar protein-protein complex and to address whether an evolutionary imperative exists for species-specific regulation of ribonucleolytic activity, RI from cow and human were produced in E. coli and purified along with their cognate ribonucleases.The binding of proteins to small ligands can increase the thermal stability of the protein through the influence of the ligand on the unfolding equilibrium of the protein (58,59,61,64). The increase in thermal stability (ΔT m ) can be related directly to the binding affinity (K d ) of ligands for the protein (75), but only when the association rate and binding enthalpy of the ligands are constant (64).…”

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confidence: 99%

Intraspecies Regulation of Ribonucleolytic Activity

2007

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The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of co-evolution higher than that for non-obligate protein-protein interactions. The coevolution of the proteins involved in certain non-obligate interactions is, however, essential to cell survival. To gain insight into the co-evolution of one such non-obligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine K d = 0.69 fM; human K d = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.The discovery of extensive protein-protein interaction networks has informed investigations of protein evolution (1-3). For example, the rate of evolution within a protein-protein interaction network, has been found to depend on the number of binding partners, the concentration of each protein, and the evolutionary age of the proteins (4-10). These relationships extend to subclasses of interactions, as obligate protein-protein interactions, which are defined as interactions necessary for the stability of an individual protein (11), evolve at a slower rate and show a greater degree of co-evolution than do non-obligate interactions, which are interactions between proteins that can remain stable independently (7). Nonetheless, the co-evolution of certain non-obligate protein-protein interactions, such as particular receptor-ligand and enzyme-inhibitor interactions, can be essential for cell survival (11). To understand the co-evolution of such a non-obligate pair, we have investigated the complex formed between the cytosolic ribonuclease inhibitor protein (RI 1 ) and secretory pancreatictype ribonucleases, as failure to inhibit the enzymatic activity of an invading ribonuclease can lead to cell death (12)(13)(14)(15) RIs (25,26), which binds to some members of the ribonuclease A superfamily with affinities in the femtomolar range (27,28). RI is able to exert this affinity using...

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Stabilization of Proteins by Ligand Binding: Application to Drug Screening and Determination of Unfolding Energetics

Cited by 165 publications

References 9 publications

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Gaining ligand selectivity in thyroid hormone receptors via entropy

Intraspecies Regulation of Ribonucleolytic Activity

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