Validation of histone-binding partners by peptide pull-downs and isothermal titration calorimetry.
In order to properly describe a chromatin-binding module and understand its biology, its binding interactions need to be specifically and explicitly defined. Tremendous gains in our understanding of the function, specificity, and concerted action of chromatin-binding complexes have been made through reductionist studies of chromatin-binding modules and posttranslationally modified histone peptides. Chromatin binding proteins often discriminate between histone posttranslational modifications and sequence contexts using subtle affinity differences that appear critical to their function. Biophysical measurements are best able to discern these minute binding energy distinctions and are increasingly important as the chromatin field endeavors to detail the unique molecular recognition of myriad chromatin states. We describe the theoretical basis and advantages of various biophysical measurements of binding affinity in the chromatin field, as well as proper experimental design and procedure for peptide pull-downs and isothermal titration calorimetry (ITC). Routine use of these techniques to characterize chromatin-binding proteins has the potential to profoundly advance our view of the molecular recognition of chromatin, allowing more quantitative comparisons across the chromatin field. Ultimately, precise determination of a binding affinity not only illuminates the biochemical and structural properties of an interface, but also informs investigation of function.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
Kathryn
K
Malecek
Alexander
A
Ruthenburg
D002151:Calorimetry; D002843:Chromatin / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006657:Histones / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D013816:Thermodynamics; D017186:Titrimetry; D054928:Validation Studies as Topic
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D055767:Immobilized Proteins; D010446:Peptide Fragments