van't Hoff enthalpies without baselines

Analysis of thermal melting curves represents one important approach for ev aluating protein stability and the consequences of amino acid substitution on protein structure. By use of the van't Hoff relationship, the differenti al melting curve can be robustly fit to only three parameters, two of which are the underlying physical constants of melting temperature (T-m) and van 't Hoff enthalpy (Delta H-nu H). Calculated T-m and Delta H-nu H values are insensitive to the choice of pre- and post-transition baselines. Consequen tly, the method accurately computes T-m and Delta H-nu H for extremely trun cated data sets, in the complete absence of baseline information, and for p roteins with low melting temperatures, where the traditional direct approac h routinely fails. Moreover, agreement between Delta H-nu H values obtained using points derived from pre- vs. post-transition data provide an indepen dent method for detecting some classes of non-two-state transitions. Finall y, fitting of the differential denaturation curve should prove useful for a nalysis of abbreviated data sets obtained from high throughput array analys is of protein stability.