SEDPHAT – A platform for global ITC analysis and global multi-method analysis of molecular interactions

Abstract

Isothermal titration calorimetry experiments can provide significantly more detailed information about molecular interactions when combined in global analysis. For example, global analysis can improve the precision of binding affinity and enthalpy, and of possible linkage parameters, even for simple bimolecular interactions, and greatly facilitate the study of multi-site and multi-component systems with competition or cooperativity. A pre-requisite for global analysis is the departure from the traditional binding model, including an ‘n’-value describing unphysical, non-integral numbers of sites. Instead, concentration correction factors can be introduced to account for either errors in the concentration determination or for the presence of inactive fractions of material. SEDPHAT is a computer program that embeds these ideas and provides a graphical user interface for the seamless combination of biophysical experiments to be globally modeled with a large number of different binding models. It offers statistical tools for the rigorous determination of parameter errors, correlations, as well as advanced statistical functions for global ITC (gITC) and global multi-method analysis (GMMA). SEDPHAT will also take full advantage of error bars of individual titration data points determined with the unbiased integration software NITPIC. The present communication reviews principles and strategies of global analysis for ITC and its extension to GMMA in SEDPHAT. We will also introduce a new graphical tool for aiding experimental design by surveying the concentration space and generating simulated data sets, which can be subsequently statistically examined for their information content. This procedure can replace the ‘c’-value as an experimental design parameter, which ceases to be helpful for multi-site systems and in the context of gITC.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

“…These kinetic binding curves could not be fit to a simple A + B ↔ AB model, suggesting a more complex association mechanism or structural heterogeneity of MHC molecules refolded with photolabile peptides. However, analysis of the equilibrium binding data with SEDPHAT (36) provided an estimate of the K d value of 4.73 μM (Cl, 3.67, 6.23), similar to the value obtained with unirradiated components by SV-AUC (1.11 μM). Remarkably, HLA-A2 complexed with photo-FluM1 58-66 , without irradiation showed rapid binding to the TAPBPR surface during the association phase, with relatively rapid dissociation during the buffer washout ( Fig.…”

A Structural Basis for Biguanide Activity

“…For competitive displacement experiments, the data for DHF binding to ecDHFR in the presence and absence of MFM were globally fit to the “Competing B and C for A” model to obtain a K d for MFM 45 .…”

Targeted Disruption of the Interaction between WD-40 Repeat Protein 5 (WDR5) and Mixed Lineage Leukemia (MLL)/SET1 Family Proteins Specifically Inhibits MLL1 and SETd1A Methyltransferase Complexes

“…Data were integrated using NITPIC (79). SEDPHAT was used to fit the integrated data to a single-site heterogeneous association model, using simulated annealing and Maxquardt-Levenberg algorithms (80). An automatic confidence interval search with projection method was applied to estimate the error of the determined thermodynamic parameters.…”