Equilibrium unfolding of the C-terminal SAM domain of p73.
The sterile alpha motif (SAM) domain is a protein module of approximately 65 to 70 amino acids found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The alpha splice variant of p73 (p73 alpha), a homologue of the tumor suppressor p53, has close to its C-terminus a SAM motif. Here, we report the folding equilibrium properties of the p73 alpha SAM domain (SAMp73) by using different biophysical techniques (circular dichroism, fluorescence, and Fourier transform infrared spectroscopies, and differential scanning calorimetry). Those probes indicate that SAMp73 folds via a two-state mechanism. Fluorescence experiments performed at different pHs showed two titrations: the first one due to an acid residue (with a pK(a) = 4.5 +/- 0.3) and the second due to deprotonation of tyrosine residues. The conformational stability of the protein upon chemical denaturation was determined over the pH range 3 to 10. The maximum conformational stability is DeltaG = 5.7 +/- 0.4 kcal x mol(-1) (at 25 degrees C) and occurs in a broad maximum, with little variation, between pH 6 and 10. The high melting temperature of SAMp73 (T(m) = 93.5 degrees C), despite its moderate conformational stability at 25 degrees C, can be ascribed to its low heat capacity change upon unfolding, DeltaC(p), which is estimated to be around 915 cal x K(-1) x mol(-1) at 25 degrees C and only around 543 cal x K(-1) x mol(-1) at the T(m). The implications of the temperature-dependent nature of DeltaC(p) are discussed in relation to the thermal stability of proteins as opposed to their conformational stability at room temperature.
Barrera
Francisco N
FN
Centro de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain.
Garzón
María T
MT
Gómez
Javier
J
Neira
José L
JL
D020816:Amino Acid Motifs; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D016147:Genes, Tumor Suppressor; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013816:Thermodynamics; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins