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Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.

ABSTRACT: The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (isobaric mass tags for relative and absolute quantification, or iTRAQ) with a covalent labeling technique, termed stability of proteins from rates of oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g., guanidine hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand-binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to the detection of both the on- and off-target effects of ligand binding. The protocol can be completed in 5 d.

SUBMITTER: Strickland EC

PROVIDER: S-EPMC3717606 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.

Strickland Erin C EC Geer M Ariel MA Tran Duc T DT Adhikari Jagat J West Graham M GM DeArmond Patrick D PD Xu Ying Y Fitzgerald Michael C MC

Nature protocols 20121220 1

The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologi ...[more]

PMID: 23257983

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Project description:Understanding how making structural changes in small molecules affects their binding affinities for targeted proteins is central to improving strategies for rational drug design. To assess the effects of varying the nature of nonpolar groups upon binding entropies and enthalpies, we designed and prepared a set of Grb2-SH2 domain ligands, Ac-pTyr-Ac6c-Asn-(CH2)n-R, in which the size and electrostatic nature of R groups at the pTyr+3 site were varied. The complexes of these ligands with the Grb2-SH2 domain were evaluated in a series of studies in which the binding thermodynamics were determined using isothermal titration calorimetry, and binding interactions were examined in crystallographic studies of two different complexes. Notably, adding nonpolar groups to the pTyr+3 site leads to higher binding affinities, but the magnitude and energetic origins of these effects vary with the nature of the R substituent. For example, enhancements to binding affinities using aliphatic R groups are driven by more favorable changes in binding entropies, whereas aryl R groups improve binding free energies through a combination of more favorable changes in binding enthalpies and entropies. However, enthalpy/entropy compensation plays a significant role in these associations and mitigates against any significant variation in binding free energies, which vary by only 0.8 kcal•mol-1, with changes in the electrostatic nature and size of the R group. Crystallographic studies show that differences in ΔG° or ΔH° correlate with buried nonpolar surface area, but they do not correlate with the total number of polar or van der Waals contacts. The relative number of ordered water molecules and relative order in the side chains at pTyr+3 correlate with differences in -TΔS°. Overall, these studies show that burial of nonpolar surface can lead to enhanced binding affinities arising from dominating entropy- or enthalpy-driven hydrophobic effects, depending upon the electrostatic nature of the apolar R group.

Dataset Information

Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.

Publications

Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.

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