Thermodynamic characterization of the folding equilibrium of the human Nedd4-WW4 domain: at the frontiers of cooperative folding.
WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.
Cobos
Eva S
ES
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 18071 Granada, Spain.
Iglesias-Bexiga
Manuel
M
Ruiz-Sanz
Javier
J
Mateo
Pedro L
PL
Luque
Irene
I
Martinez
Jose C
JC
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D000075702:Nedd4 Ubiquitin Protein Ligases; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
D056827:Endosomal Sorting Complexes Required for Transport; D007527:Isoenzymes; D014364:Tryptophan; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases